Recombinant thermostable enzyme for converting maltose into trehalose from Thermus aquaticus

ABSTRACT

Disclosed are a recombinant thermostable enzyme, which converts maltose into trehalose and is stable up to a temperature of about 80 DEG  C. even when incubated at pH 7.0 for 60 min, a preparation of the enzyme, a DNA encoding the enzyme, a recombinant DNA containing the DNA, a transformant, and an enzymatic conversion method of maltose by using the enzyme.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel recombinant thermostable enzyme which converts maltose into trehalose.

2. Description of the Prior Art

Trehalose is a disaccharide which consists of 2 glucose molecules linked together with their reducing groups, and, naturally, it is present in bacteria, fungi, algae, insects, etc., in an extremely-small quantity. Having no reducing residue within the molecule, trehalose does not cause an unsatisfactory browning reaction even when heated in the presence of amino acids or the like, and because of this it can advantageously sweeten food products without fear of causing unsatisfactory coloration and deterioration. However, trehalose is far from being readily prepared in a desired amount by conventional methods, and, actually, it is not scarcely used for sweetening food products.

Conventional methods are roughly classified into 2 groups, i.e. the one using cells of microorganisms and the other employing a multi-enzymatic system wherein enzymes are allowed to act on saccharides. The former, as disclosed in Japanese Patent Laid-Open No. 154,485/75, is a method which comprises allowing to grow microorganisms such as bacteria and yeasts in a nutrient culture medium, and collecting trehalose from the resultant culture. The latter, as disclosed in Japanese Patent Laid-Open No. 216,695/83, is a method which comprises providing maltose as a substrate, allowing a multi-enzymatic system using maltose- and trehalose-phosphorylases to act on maltose, and isolating the formed trehalose from the reaction system. Although the former facilitates the growth of microorganisms without special difficulty, it has a drawback that the resultant culture only contains at most 15 w/w % trehalose, on a dry solid basis (d.s.b.). While the latter enables the separation of trehalose with a relative easiness, but it is theoretically difficult to increase the trehalose yield by allowing enzymes to act on substrates at a considerably-high concentration because the enzymatic reaction per se is an equilibrium reaction of 2 different types of enzymes and the equilibrium point constantly inclines to the side of forming glucose phosphate.

In view of the foregoing, the present inventors energetically screened enzymes which directly convert maltose into trehalose, and have found that microorganisms belonging to those of the genera Pimelobacter and Pseudomonas, as disclosed in Japanese Patent Application No. 199,971/93, produce an absolutely novel enzyme which forms trehalose when acts on maltose. This means that trehalose can be prepared from maltose as a material which is readily available in quantity and at low cost, and the use of the enzyme would completely overcome all the aforesaid objects.

It was found that all the enzymes from these microorganisms have an optimum temperature of about 20°-40° C. which seems some how insufficient for trehalose production in their thermostability. It is recognized in this field that the saccharification of starch and amylaceous substances should be generally reacted at a temperature of over 55° C.: If the saccharification reaction is effected at a temperature of 55° C. or lower, bacterial contamination is enhanced to lower the pH of the reaction mixtures and to inactivate enzymes used, followed by remaining a relatively large amount of substrates intact. If the saccharification reaction is effected by using enzymes with poor thermostability, a great care should be taken for the pH changes, and, once a pH lowering occurs, alkalis should be quickly added to the reaction mixtures to increase the pH.

In view of the foregoing, the present inventors further studied on thermostable enzymes with such activity and have found that enzymes, produced from microorganisms of the genus Thermus such as a microorganism of the species Thermus aquaticus (ATCC 33923), effectively convert maltose into trehalose without being substantially inactivated even when reacted at a temperature of over 55° C. These enzymes, however, are not sufficient in enzyme producing activity, and this leads to a problem of that an industrial scale production of trehalose inevitably requires a considerably large scale cultivation of such microorganisms.

Recombinant DNA technology has made a remarkable progress in recent years. At present, even an enzyme, whose total amino acid sequence is not revealed, can be readily prepared in a desired amount, if a gene encoding the enzyme was once isolated and the base sequence was decoded, by preparing a recombinant DNA containing a DNA which encodes the enzyme, introducing the recombinant DNA into microorganisms or cells of plants or animals, and culturing the resultant transformants. Under these circumstances, urgently required are to find a gene encoding the above thermostable enzyme and to decode the base sequence.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a recombinant thermostable enzyme which forms trehalose when acts on maltose.

It is a further object of the present invention to provide a DNA which encodes the recombinant enzyme.

It is yet another object of the present invention to provide a replicable recombinant DNA having the DNA.

It is a further object of the present invention to provide a transformant into which the recombinant DNA has been introduced.

It is a further object of the present invention to provide a process for preparing the recombinant enzyme by using the transformant.

It is a further object of the present invention to provide a method for converting maltose into trehalose by the recombinant enzyme.

Means to Attain the Object!

The first object of the present invention is attained by a recombinant enzyme.

The second object of the present invention is attained by a DNA which encodes the recombinant enzyme.

The third object of the present invention is attained by a replicable recombinant DNA which contains the DNA and a self-replicable vector.

The fourth object of the present invention is attained by a transformant obtained by introducing the replicable recombinant DNA into an appropriate host.

The fifth object of the present invention is attained by culturing the transformant in a nutrient culture medium to form the recombinant enzyme, and collecting the formed recombinant enzyme from the resultant culture.

The sixth object of the present invention is attained by an enzymatic conversion method of maltose which contains a step of allowing the recombinant enzyme to act on maltose to form trehalose.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG.1 shows the optimum temperature of an enzyme produced from Thermus aquaticus (ATCC 33923).

FIG.2 shows the optimum pH of an enzyme produced from Thermus aquaticus (ATCC 33923).

FIG.3 shows the thermal stability of an enzyme produced from Thermus aquaticus (ATCC 33923).

FIG.4 shows the pH stability of an enzyme produced from Thermus aquaticus (ATCC 33923).

FIG.5 shows the structure of the recombinant DNA pBTM22 according to the present invention.

FIG.6 shows the structure of the recombinant DNA pBTM23 according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The recombinant enzyme according to the present invention acts on maltose to form trehalose without being substantially inactivated even when allowed to react at a temperature of over 55° C.

The DNA according to the present invention expresses the production of the present recombinant enzyme when introduced into an appropriate self-replicable vector to obtain a replicable recombinant DNA, then introduced into an appropriate host, which is inherently incapable of forming the recombinant enzyme but readily proliferative, to form a transformant.

The recombinant DNA according to the present invention expresses the production of the recombinant enzyme by introducing it into an appropriate host, which is inherently incapable of forming the recombinant enzyme but readily proliferative, to form a transformant, and culturing the transformant in a nutrient culture medium.

The transformant forms a desired amount of the recombinant enzyme when cultured according to the present invention.

The enzymatic conversion method according to the present invention converts maltose into a saccharide composition comprising trehalose, glucose and/or maltooligosaccharides.

The present invention was made based on the finding of an absolutely novel thermostable enzyme which converts maltose into trehalose. Such an enzyme can be obtained from cultures of Thermus aquaticus (ATCC 33923), and the present inventors isolated the enzyme by using a variety of methods comprising column chromatography as a main technique, and studied on the properties and features, revealing that the reality is a polypeptide having the following physicochemical properties:

(1) Action Forming trehalose when acts on maltose, and vice versa;

(2) Molecular weight (MW) About 100,000-110,000 daltons when assayed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE);

(3) Isoelectric point (pI) About 3.8-4.8 when assayed on isoelectrophoresis;

(4) Optimum temperature About 65° C. when incubated at pH 7.0 for 60 min;

(5) Optimum pH About 6.0-6.7 when incubated at 60° C. for 60 min;

(6) Thermal stability Stable up to a temperature of about 80° C. even when incubated at pH 7.0 for 60 min; and

(7) pH Stability Stable up to a pH of 5.5-9.5 even when incubated at 60° C. for 60 min.

Experiments for revealing the physicochemical properties of a thermostable enzyme produced from Thermus aquaticus (ATCC 33923) are as follows:

EXPERIMENT 1 PURIFICATION OF ENZYME

Experiment 1-1

Production of enzyme

In 500-ml Erlenmeyer flasks were placed 100 ml aliquots of a liquid culture medium (pH 7.5) containing 0.5 w/v % polypeptone, 0.1 w/v % yeast extract, 0.07 w/v % sodium nitrate, 0.01 w/v % disodium hydrogen phosphate, 0.02 w/v % magnesium sulfate heptahydrate, 0.01 w/v % calcium chloride, and water, and the flasks were autoclaved at 120° C. for 20 min to effect sterilization. After cooling the flasks a seed culture of Thermus aquaticus (ATCC 33923) was inoculated into each flask, followed by the incubation at 60° C. for 24 hours under a rotary-shaking condition of 200 rpm to obtain a seed culture. Twenty L aliquots of a fresh preparation of the same liquid culture medium were put in 30-L jar fermenters, sterilized and cooled to 60° C., followed by inoculating one v/v % of the seed culture into each fermenter, and incubating the resultant at a pH of 6.0-8.0 and 60° C. for about 20 hours under aeration-agitation conditions.

Thereafter, the enzymatic activity of the resultant culture was assayed to reveal that it contained about 0.35 units/ml of the enzyme. A portion of the culture was centrifuged, and the supernatant was assayed to reveal that it contained about 0.02 units/ml of the enzyme. While the separated cells were suspended in 50 mM phosphate buffer (pH 7.0) to give the total volume equal to the original volume of the portion, followed by assaying the suspension to reveal that it contained about 0.33 units/ml of the enzyme.

Throughout the specification the enzyme activity is expressed by the value measured on the following assay: Place one ml of 10 mM phosphate buffer (pH 7.0) containing 20 w/v % maltose in a test tube, add one ml of an appropriately diluted enzyme solution to the tube, and incubate the solution in the tube at 60° C. for 60 min to effect an enzymatic reaction, followed by a further incubation at 100° C. for 10 min to suspend the enzymatic reaction. Thereafter, a portion of the reaction mixture was diluted by 11 times with 50 mM phosphate buffer (pH 7.5), and 0.4 ml of which was placed in a test tube, admixed with 0.1 ml solution containing one unit/ml trehalase, followed by incubating the resultant mixture at 45° C. for 120 min and quantifying the glucose content on the glucose oxidase method. As a control, a system using a trehalase solution and an enzyme solution which has been inactivated by heating at 100° C. for 10 min is provided and treated similarly as above. The content of the formed trehalose is estimable based on the content of glucose quantified in the above. One unit of the enzyme activity is defined as the amount which forms one umol trehalose per min under the above conditions.

Experiment 1-2

Purification of enzyme

The culture obtained in Experiment 1-1 was centrifuged to separate cells, and about 0.28 kg of the wet cells thus obtained was suspended in 10 mM phosphate buffer (pH 7.0), disrupted in usual manner, and centrifuged to obtain an about 1.8 L of a crude enzyme solution. The solution was admixed with ammonium sulfate to give a saturation of 70 w/v %, salted out by standing at 4° C. overnight, and centrifuged to obtain a supernatant. The supernatant was mixed with 10 mM phosphate buffer (pH 7.0), and the mixture solution was dialyzed against a fresh preparation of the same buffer for 24 hours.

The dialyzed inner solution was centrifuged to obtain a supernatant (1,560 ml) which was then applied to a column packed with 530 ml of "DEAE-TOYOPEARL® 650", an ion exchanger commercialized by Tosoh Corporation, Tokyo, Japan, which had been previously equilibrated with 10 mM phosphate buffer (pH 7.0), followed by feeding to the column a linear gradient buffer of sodium chloride ranging from 0M to 0.4M in 10 mM phosphate buffer (pH 7.0). From the eluate, fractions with the objective enzyme activity were collected, pooled, dialyzed against 10 mM phosphate buffer (pH 7.0) containing one M ammonium sulfate for 10 hours, and centrifuged to obtain a supernatant. The supernatant was applied to a column packed with 380 ml of "BUTYL-TOYOPEARL® 650", a gel for hydrophobic chromatography commercialized by Tosoh Corporation, Tokyo, Japan, which had been previously equilibrated with 10 mM phosphate buffer (pH 7.0) containing one M ammonium sulfate, followed by feeding to the column a linear gradient buffer of ammonium sulfate ranging from 1M to 0M in 10 mM phosphate buffer (pH 7.0).

Fractions, eluted at 0.2M ammonium sulfate, with the objective enzyme activity were collected, pooled and dialyzed against 10 mM phosphate buffer (pH 7.0) containing 0.2M sodium chloride for 16 hours. The dialyzed solution was centrifuged to remove insoluble substances, fed to a column packed with 380 ml of "TOYOPEARL® HW-55S", a gel for gel filtration chromatography commercialized by Tosoh, Corporation, Tokyo, Japan, which had been previously equilibrated with 10 mM phosphate buffer (pH 7.0) containing 0.2M sodium chloride, followed by feeding to the column with 10 mM phosphate buffer (pH 7.0) containing one M sodium chloride. Fractions with the enzyme activity were collected from the eluate, fed to a column packed with "MONO Q HR5/5" which had been equilibrated with 10 mM phosphate buffer (pH 7.0). The column was fed with a linear gradient buffer of sodium chloride ranging from 0.1M to 0.35M in 10 mM phosphate buffer (pH 7.0), followed by collecting fractions with the enzyme activity. The purified enzyme thus obtained had a specific activity of about 135 units/mg protein in a yield of about 330 units per L of the culture.

The purified enzyme was electrophoresed in a 7.5 w/v % polyacrylamide gel to give a single protein band with the enzyme activity, and this meant that it had a considerably-high purity.

EXPERIMENT 2 PHYSICOCHEMICAL PROPERTY OF ENZYME

Experiment 2-1

Action

To an aqueous solution containing 5 w/w % maltose or trehalose as a substrate was added 2 units/g substrate of the purified enzyme obtained in Experiment 1-2, and the mixture was incubated at 60° C. and pH 7.0 for 24 hours. In order to analyze the saccharide composition of the reaction mixture, it was dried in vacuo, dissolved in pyridine, and trimethylsilylated in usual manner, and the resultant was subjected to gas chromatography. The equipments and conditions used in this analysis were as follows: "GC-16A" commercialized by Shimadzu Seisakusho, Ltd., Tokyo, Japan, as a gas chromatograph; a stainless steel column, having an inner diameter of 3 mm and a length of 2 m, packed with 2% "SILICONE OV-17/CHROMOSOLB W" commercialized by GL Sciences Inc., Tokyo, Japan, as a column; a hydrogen flame type of ionization as a detector; nitrogen gas as a carrier gas (flow rate of 40 ml/min); and a column oven temperature of 160°-320° C. at a programmed increasing temperature rate of 7.5° C./min. The saccharide compositions of the reaction mixtures were tabulated in Table 1:

                  TABLE 1                                                          ______________________________________                                                 Saccharide composition of                                                      reaction mixture (%)                                                   Substrate Trehalose     Glucose Maltose                                        ______________________________________                                         Maltose   70.0          4.4     25.6                                           Trehalose 76.2          3.1     20.7                                           ______________________________________                                    

As is shown in Table 1, the purified enzyme formed about 70 w/w % trehalose and about 4 w/w % glucose when acted on maltose as a substrate, while it formed about 21 w/w % maltose and about 3 w/w % glucose when acted on trehalose as a substrate. These facts indicate that the purified enzyme has activities of converting maltose into trehalose and of converting trehalose into maltose, as well as of hydrolyzing α-1,4 linkage in maltose molecule and α,α-1,1 linkage in trehalose molecule. There has been no report of such an enzyme, and this leads to an estimation of having a novel enzymatic pathway.

Experiment 2-2

Molecular weight

In accordance with the method as reported by U. K. Laemmli in Nature, Vol.227, pp.680-685 (1970), the purified enzyme was electrophoresed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to give a single protein band at a position corresponding to about 100,000-110,000 daltons. The marker proteins used in this experiment were myosin (MW=200,000 daltons), β-galactosidase (MW=116,250 daltons), phosphorylase B (MW=97,400 daltons), serum albumin (MW=66,200 daltons) and ovalbumin (MW=45,000 daltons).

Experiment 2-3

Isoelectric point

The purified enzyme gave an isoelectric point of about 3.8-4.8 when isoelectrophoresed in 2 w/v % "AMPHOLINE®", a polyacrylamide gel commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden.

Experiment 2-4

Optimum temperature

The optimum temperature of the purified enzyme was about 65° C. as shown in FIG. 1 when incubated in usual manner in 10 mM phosphate buffer (pH 7.0) for 60 min.

Experiment 2-5

Optimum pH

The optimum pH of the purified enzyme was about 6.0-6.7 as shown in FIG. 2 when tested in usual manner by incubating it at 60° C. for 60 min in 10 mM acetate buffer, phosphate buffer or sodium carbonate/sodium hydrogen carbonate buffer with different pHs.

Experiment 2-6

Thermal stability

The purified enzyme was stable up to a temperature of about 80° C. as shown in FIG. 3 when tested in usual manner by incubating it in 50 mM phosphate buffer (pH 7.0) for 60 min.

Experiment 2-7

pH Stability

The purified enzyme was stable up to a pH of about 5.5-9.5 as shown in FIG. 4 when experimented in usual manner by incubating it at 60° C. for 60 min in 50 mM acetate buffer, phosphate buffer or sodium carbonate/sodium hydrogen carbonate buffer with different pHs.

Experiment 2-8

Amino acid sequence containing the N-terminus

The amino acid sequence containing the N-terminus of the purified enzyme was analyzed on "MODEL 470A", a gas-phase protein sequencer commercialized by Perkin-Elmer Corp., Instrument Div., Norrwalk, USA, and revealed to have the amino acid sequence containing the N-terminus in SEQ ID NO:1. ##STR1## Experiment 2-9

Partial amino acid sequence

An adequate amount of the purified enzyme prepared in Experiment 1-2 was weighed, dialyzed against 10 mM Tris-HCl buffer (pH 9.0) at 4° C. for 18 hours, and admixed with 10 mM Tris-HCl buffer (pH 9.0) to obtain a solution containing about one mg/ml of the enzyme. The solution was incubated at 100° C. for 5 min to denature the enzyme, and about one ml of which was placed in a test tube, admixed with 40 μg lysyl endopeptidase, and incubated at 30° C. for 44 hours to partially hydrolyze the enzyme. The resultant hydrolysate was applied to "μBONDASPERE C18", a column for reverse-phase high-performance liquid chromatography commercialized by Japan Millipore Ltd., Tokyo, Japan, which had been equilibrated with 0.1 v/v % trifluoroacetate, followed by feeding to the column 0.1 v/v % trifluoroacetate containing acetonitrile at a flow rate of 1.0 ml/min while increasing the concentration of acetonitrile from 0 v/v % to 70 v/v %.

Fractions containing a peptide fragment eluted about 58 min to 60 min after the initiation of the feeding were collected, pooled, dried in vacuo, and dissolved in 0.5 ml of 10 mM Tris-HCl buffer (pH 8.0), admixed with 5 μg TPCK treated trypsin, and incubated at 37° C. for 16 hours to effect hydrolysis. The enzymatic reaction was suspended by freezing, and the resultant hydrolyzate was fed to a column packed with "μBONDASPERE C18", followed by feeding to the column 0.1 v/v % trifluoroacetate containing aqueous acetonitrile at a flow rate of 1.0 ml/min while increasing the concentration of aqueous acetonitrile from 15 v/v % to 55 v/v %. Fractions, containing a peptide fragment eluted about 42 min after the initiation of the feeding, were collected, pooled, dried in vacuo, and dissolved in 0.1 v/v trifluoroacetate containing 50 v/v % aqueous acetonitrile. Similarly as in Experiment 2-8, it was revealed that the peptide fragment contained the amino acid sequence in SEQ ID NO:2. ##STR2##

Since no enzyme with these physicochemical properties has been known, it can be estimated to be a novel substance.

The present inventors energetically screened the chromosomal DNA of Thermus aquaticus (ATCC 33923) by using an oligonucleotide as a probe which had been chemically synthesized based on the amino acid sequences as revealed in Experiments 2-8 and 2-9, and have obtained a DNA fragment which consisted of about 3,600 base pairs having the base sequence in SEQ ID NO:4. The decoding of the base sequence revealed that a thermostable enzyme from the microorganism consists of 963 amino acids and has the amino acid sequence in SEQ ID NO:3.

    __________________________________________________________________________     SEQ ID NO:3:                                                                   Met                                                                               Asp                                                                               Pro Leu                                                                               Trp                                                                               Tyr                                                                               Lys Asp                                                                               Ala                                                                               Val                                                                               Ile Tyr                                                                               Gln                                                                               Leu                                                                               His                                                                               Val                            1            5               10              15                                Arg                                                                               Ser                                                                               Phe Phe                                                                               Asp                                                                               Ala                                                                               Asn Asn                                                                               Asp                                                                               Gly                                                                               Tyr Gly                                                                               Asp                                                                               Phe                                                                               Glu                                                                               Gly                                      20              25              30                                   Leu                                                                               Arg                                                                               Arg Lys                                                                               Leu                                                                               Pro                                                                               Tyr Leu                                                                               Glu                                                                               Glu                                                                               Leu Gly                                                                               Val                                                                               Asn                                                                               Thr                                                                               Leu                                  35               40              45                                      Trp                                                                               Leu                                                                               Met Pro                                                                               Phe                                                                               Phe                                                                               Gln Ser                                                                               Pro                                                                               Leu                                                                               Arg Asp                                                                               Asp                                                                               Gly                                                                               Tyr                                                                               Asp                               50              55               60                                         Ile                                                                               Ser                                                                               Asp Tyr                                                                               Tyr                                                                               Gln                                                                               Ile Leu                                                                               Pro                                                                               Val                                                                               His Gly                                                                               Thr                                                                               Leu                                                                               Glu                                                                               Asp                            65              70              75              80                             Phe                                                                               Thr                                                                               Val Asp                                                                               Glu                                                                               Ala                                                                               His Gly                                                                               Arg                                                                               Gly                                                                               Met Lys                                                                               Val                                                                               Ile                                                                               Ile                                                                               Glu                                         85              90              95                                Leu                                                                               Val                                                                               Leu Asn                                                                               His                                                                               Thr                                                                               Ser Ile                                                                               Asp                                                                               His                                                                               Pro Trp                                                                               Phe                                                                               Gln                                                                               Glu                                                                               Ala                                      100             105             110                                  Arg                                                                               Lys                                                                               Pro Asn                                                                               Ser                                                                               Pro                                                                               Met Arg                                                                               Asp                                                                               Trp                                                                               Tyr Val                                                                               Trp                                                                               Ser                                                                               Asp                                                                               Thr                                  115              120             125                                     Pro                                                                               Glu                                                                               Lys Tyr                                                                               Lys                                                                               Gly                                                                               Val Arg                                                                               Val                                                                               Ile                                                                               Phe Lys                                                                               Asp                                                                               Phe                                                                               Glu                                                                               Thr                               130             135              140                                        Ser                                                                               Asn                                                                               Trp Thr                                                                               Phe                                                                               Asp                                                                               Pro Val                                                                               Ala                                                                               Lys                                                                               Ala Tyr                                                                               Tyr                                                                               Trp                                                                               His                                                                               Arg                            145             150             155             160                            Phe                                                                               Tyr                                                                               Trp His                                                                               Gln                                                                               Pro                                                                               Asp Leu                                                                               Asn                                                                               Trp                                                                               Asp Ser                                                                               Pro                                                                               Glu                                                                               Val                                                                               Glu                                         165             170             175                               Lys                                                                               Ala                                                                               Ile His                                                                               Gln                                                                               Val                                                                               Met Phe                                                                               Phe                                                                               Trp                                                                               Ala Asp                                                                               Leu                                                                               Gly                                                                               Val                                                                               Asp                                      180             185             190                                  Gly                                                                               Phe                                                                               Arg Leu                                                                               Asp                                                                               Ala                                                                               Ile Pro                                                                               Tyr                                                                               Leu                                                                               Tyr Glu                                                                               Arg                                                                               Glu                                                                               Gly                                                                               Thr                                  195              200             205                                     Ser                                                                               Cys                                                                               Glu Asn                                                                               Leu                                                                               Pro                                                                               Glu Thr                                                                               Ile                                                                               Glu                                                                               Ala Val                                                                               Lys                                                                               Arg                                                                               Leu                                                                               Arg                               210             215              220                                        Lys                                                                               Ala                                                                               Leu Glu                                                                               Glu                                                                               Arg                                                                               Tyr Gly                                                                               Pro                                                                               Gly                                                                               Lys Ile                                                                               Leu                                                                               Leu                                                                               Ala                                                                               Glu                            225             230             235             240                            Ala                                                                               Asn                                                                               Met Trp                                                                               Pro                                                                               Glu                                                                               Glu Thr                                                                               Leu                                                                               Pro                                                                               Tyr Phe                                                                               Gly                                                                               Asp                                                                               Gly                                                                               Asp                                         245             250             255                               Gly                                                                               Val                                                                               His Met                                                                               Ala                                                                               Tyr                                                                               Asn Phe                                                                               Pro                                                                               Leu                                                                               Met Pro                                                                               Arg                                                                               Ile                                                                               Phe                                                                               Met                                      260             265             270                                  Ala                                                                               Leu                                                                               Arg Arg                                                                               Glu                                                                               Asp                                                                               Arg Gly                                                                               Pro                                                                               Ile                                                                               Glu Thr                                                                               Met                                                                               Leu                                                                               Lys                                                                               Glu                                  275              280             285                                     Ala                                                                               Glu                                                                               Gly Ile                                                                               Pro                                                                               Glu                                                                               Thr Ala                                                                               Gln                                                                               Trp                                                                               Ala Leu                                                                               Phe                                                                               Leu                                                                               Arg                                                                               Asn                               290             295              300                                        His                                                                               Asp                                                                               Glu Leu                                                                               Thr                                                                               Leu                                                                               Glu Lys                                                                               Val                                                                               Thr                                                                               Glu Glu                                                                               Glu                                                                               Arg                                                                               Glu                                                                               Phe                            305             310             315             320                            Met                                                                               Tyr                                                                               Glu Ala                                                                               Tyr                                                                               Ala                                                                               Pro Asp                                                                               Pro                                                                               Lys                                                                               Phe Arg                                                                               Ile                                                                               Asn                                                                               Leu                                                                               Gly                                         325             330             335                               Ile                                                                               Arg                                                                               Arg Arg                                                                               Leu                                                                               Met                                                                               Pro Leu                                                                               Leu                                                                               Gly                                                                               Gly Asp                                                                               Arg                                                                               Arg                                                                               Arg                                                                               Tyr                                      340             345             350                                  Glu                                                                               Leu                                                                               Leu Thr                                                                               Ala                                                                               Leu                                                                               Leu Leu                                                                               Thr                                                                               Leu                                                                               Lys Gly                                                                               Thr                                                                               Pro                                                                               Ile                                                                               Val                                  355              360             365                                     Tyr                                                                               Tyr                                                                               Gly Asp                                                                               Glu                                                                               Ile                                                                               Gly Met                                                                               Gly                                                                               Asp                                                                               Asn Pro                                                                               Phe                                                                               Leu                                                                               Gly                                                                               Asp                               370             375              380                                        Arg                                                                               Asn                                                                               Gly Val                                                                               Arg                                                                               Thr                                                                               Pro Met                                                                               Gln                                                                               Trp                                                                               Ser Gln                                                                               Asp                                                                               Arg                                                                               Ile                                                                               Val                            385             390             395             400                            Ala                                                                               Phe                                                                               Ser Arg                                                                               Ala                                                                               Pro                                                                               Tyr His                                                                               Ala                                                                               Leu                                                                               Phe Leu                                                                               Pro                                                                               Pro                                                                               Val                                                                               Ser                                         405             410             415                               Glu                                                                               Gly                                                                               Pro Tyr                                                                               Ser                                                                               Tyr                                                                               His Phe                                                                               Val                                                                               Asn                                                                               Val Glu                                                                               Ala                                                                               Gln                                                                               Arg                                                                               Glu                                      420             425             430                                  Asn                                                                               Pro                                                                               His Ser                                                                               Leu                                                                               Leu                                                                               Ser Phe                                                                               Asn                                                                               Arg                                                                               Arg Phe                                                                               Leu                                                                               Ala                                                                               Leu                                                                               Arg                                  435              440             445                                     Asn                                                                               Gln                                                                               His Ala                                                                               Lys                                                                               Ile                                                                               Phe Gly                                                                               Arg                                                                               Gly                                                                               Ser Leu                                                                               Thr                                                                               Leu                                                                               Leu                                                                               Pro                               450             455              460                                        Val                                                                               Glu                                                                               Asn Arg                                                                               Arg                                                                               Val                                                                               Leu Ala                                                                               Tyr                                                                               Leu                                                                               Arg Glu                                                                               His                                                                               Glu                                                                               Gly                                                                               Glu                            465             470             475             480                            Arg                                                                               Val                                                                               Leu Val                                                                               Val                                                                               Ala                                                                               Asn Leu                                                                               Ser                                                                               Arg                                                                               Tyr Thr                                                                               Gln                                                                               Ala                                                                               Phe                                                                               Asp                                         485             490             495                               Leu                                                                               Pro                                                                               Leu Glu                                                                               Ala                                                                               Tyr                                                                               Gln Gly                                                                               Leu                                                                               Val                                                                               Pro Val                                                                               Glu                                                                               Leu                                                                               Phe                                                                               Ser                                      500             505             510                                  Gln                                                                               Gln                                                                               Pro Phe                                                                               Pro                                                                               Pro                                                                               Val Glu                                                                               Gly                                                                               Arg                                                                               Tyr Arg                                                                               Leu                                                                               Thr                                                                               Leu                                                                               Gly                                  515              520             525                                     Pro                                                                               His                                                                               Gly Phe                                                                               Ala                                                                               Leu                                                                               Phe Ala                                                                               Leu                                                                               Lys                                                                               Pro Val                                                                               Glu                                                                               Ala                                                                               Val                                                                               Leu                               530             535              540                                        His                                                                               Leu                                                                               Pro Ser                                                                               Pro                                                                               Asp                                                                               Trp Ala                                                                               Glu                                                                               Glu                                                                               Pro Ala                                                                               Pro                                                                               Glu                                                                               Glu                                                                               Ala                            545             550             555             560                            Asp                                                                               Leu                                                                               Pro Arg                                                                               Val                                                                               His                                                                               Met Pro                                                                               Gly                                                                               Gly                                                                               Pro Glu                                                                               Val                                                                               Leu                                                                               Leu                                                                               Val                                         565             570             575                               Asp                                                                               Thr                                                                               Leu Val                                                                               His                                                                               Glu                                                                               Arg Gly                                                                               Arg                                                                               Glu                                                                               Glu Leu                                                                               Leu                                                                               Asn                                                                               Ala                                                                               Leu                                      580             585             590                                  Ala                                                                               Gln                                                                               Thr Leu                                                                               Lys                                                                               Glu                                                                               Lys Ser                                                                               Trp                                                                               Leu                                                                               Ala Leu                                                                               Lys                                                                               Pro                                                                               Gln                                                                               Lys                                  595              600             605                                     Val                                                                               Ala                                                                               Leu Leu                                                                               Asp                                                                               Ala                                                                               Leu Arg                                                                               Phe                                                                               Gln                                                                               Lys Asp                                                                               Pro                                                                               Pro                                                                               Leu                                                                               Tyr                               610             615              620                                        Leu                                                                               Thr                                                                               Leu Leu                                                                               Gln                                                                               Leu                                                                               Glu Asn                                                                               His                                                                               Arg                                                                               Thr Leu                                                                               Gln                                                                               Val                                                                               Ser                                                                               Leu                            625             630             635             640                            Pro                                                                               Leu                                                                               Leu Trp                                                                               Ser                                                                               Pro                                                                               Gln Arg                                                                               Arg                                                                               Glu                                                                               Gly Pro                                                                               Gly                                                                               Leu                                                                               Phe                                                                               Ala                                         645             650             655                               Arg                                                                               Thr                                                                               His Gly                                                                               Gln                                                                               Pro                                                                               Gly Tyr                                                                               Phe                                                                               Tyr                                                                               Glu Leu                                                                               Ser                                                                               Leu                                                                               Asp                                                                               Pro                                      660             665             670                                  Gly                                                                               Phe                                                                               Tyr Arg                                                                               Leu                                                                               Leu                                                                               Leu Ala                                                                               Arg                                                                               Leu                                                                               Lys Glu                                                                               Gly                                                                               Phe                                                                               Glu                                                                               Gly                                  675              680             685                                     Arg                                                                               Ser                                                                               Leu Arg                                                                               Ala                                                                               Tyr                                                                               Tyr Arg                                                                               Gly                                                                               Arg                                                                               His Pro                                                                               Gly                                                                               Pro                                                                               Val                                                                               Pro                               690             695              700                                        Glu                                                                               Ala                                                                               Val Asp                                                                               Leu                                                                               Leu                                                                               Arg Pro                                                                               Gly                                                                               Leu                                                                               Ala Ala                                                                               Gly                                                                               Glu                                                                               Gly                                                                               Val                            705             710             715             720                            Trp                                                                               Val                                                                               Gln Leu                                                                               Gly                                                                               Leu                                                                               Val Gln                                                                               Asp                                                                               Gly                                                                               Gly Leu                                                                               Asp                                                                               Arg                                                                               Thr                                                                               Glu                                         725             730             735                               Arg                                                                               Val                                                                               Leu Pro                                                                               Arg                                                                               Leu                                                                               Asp Leu                                                                               Pro                                                                               Trp                                                                               Val Leu                                                                               Arg                                                                               Pro                                                                               Glu                                                                               Gly                                      740             745             750                                  Gly                                                                               Leu                                                                               Phe Trp                                                                               Glu                                                                               Arg                                                                               Gly Ala                                                                               Ser                                                                               Arg                                                                               Arg Val                                                                               Leu                                                                               Ala                                                                               Leu                                                                               Thr                                  755              760             765                                     Gly                                                                               Ser                                                                               Leu Pro                                                                               Pro                                                                               Gly                                                                               Arg Pro                                                                               Gln                                                                               Asp                                                                               Leu Phe                                                                               Ala                                                                               Ala                                                                               Leu                                                                               Glu                               770             775              780                                        Val                                                                               Arg                                                                               Leu Leu                                                                               Glu                                                                               Ser                                                                               Leu Pro                                                                               Arg                                                                               Leu                                                                               Arg Gly                                                                               His                                                                               Ala                                                                               Pro                                                                               Gly                            785             790             795             800                            Thr                                                                               Pro                                                                               Gly Leu                                                                               Leu                                                                               Pro                                                                               Gly Ala                                                                               Leu                                                                               His                                                                               Glu Thr                                                                               Glu                                                                               Ala                                                                               Leu                                                                               Val                                         805             810             815                               Arg                                                                               Leu                                                                               Leu Gly                                                                               Val                                                                               Arg                                                                               Leu Ala                                                                               Leu                                                                               Leu                                                                               His Arg                                                                               Ala                                                                               Leu                                                                               Gly                                                                               Glu                                      820             825             830                                  Val                                                                               Glu                                                                               Gly Val                                                                               Val                                                                               Gly                                                                               Gly His                                                                               Pro                                                                               Leu                                                                               Leu Gly                                                                               Arg                                                                               Gly                                                                               Leu                                                                               Gly                                  835              840             845                                     Ala                                                                               Phe                                                                               Leu Glu                                                                               Leu                                                                               Glu                                                                               Gly Glu                                                                               Val                                                                               Tyr                                                                               Leu Val                                                                               Ala                                                                               Leu                                                                               Gly                                                                               Ala                               850             855              860                                        Glu                                                                               Lys                                                                               Arg Gly                                                                               Thr                                                                               Val                                                                               Glu Glu                                                                               Asp                                                                               Leu                                                                               Ala Arg                                                                               Leu                                                                               Ala                                                                               Tyr                                                                               Asp                            865             870             875             880                            Val                                                                               Glu                                                                               Arg Ala                                                                               Val                                                                               His                                                                               Leu Ala                                                                               Leu                                                                               Glu                                                                               Ala Leu                                                                               Glu                                                                               Ala                                                                               Glu                                                                               Leu                                         885             890             895                               Trp                                                                               Ala                                                                               Phe Ala                                                                               Glu                                                                               Glu                                                                               Val Ala                                                                               Asp                                                                               His                                                                               Leu His                                                                               Ala                                                                               Ala                                                                               Phe                                                                               Leu                                      900             905             910                                  Gln                                                                               Ala                                                                               Tyr Arg                                                                               Ser                                                                               Ala                                                                               Leu Pro                                                                               Glu                                                                               Glu                                                                               Ala Leu                                                                               Glu                                                                               Glu                                                                               Ala                                                                               Gly                                  915              920             925                                     Trp                                                                               Thr                                                                               Arg His                                                                               Met                                                                               Ala                                                                               Glu Val                                                                               Ala                                                                               Ala                                                                               Glu His                                                                               Leu                                                                               His                                                                               Arg                                                                               Glu                               930             935              940                                        Glu                                                                               Arg                                                                               Pro Ala                                                                               Arg                                                                               Lys                                                                               Arg Ile                                                                               His                                                                               Glu                                                                               Arg Trp                                                                               Gln                                                                               Ala                                                                               Lys                                                                               Ala                            945             950             955             960                            Gly                                                                               Lys                                                                               Ala                                                                      SEQ ID NO:4:                                                                   GTGGACCCCC                                                                             TCTGGTACAA                                                                             GGACGCGGTG                                                                             ATCTACCAGC                                                                             TCCACGTCCG                                                                             CTCCTTCTTT                                                                              60                            GACGCCAACA                                                                             ACGACGGCTA                                                                             CGGGGACTTT                                                                             GAGGGCCTGA                                                                             GGCGGAAGCT                                                                             TCCCTACCTG                                                                              120                           GAGGAGCTCG                                                                             GGGTCAACAC                                                                             CCTCTGGCTC                                                                             ATGCCCTTCT                                                                             TCCAGTCCCC                                                                             CTTGAGGGAC                                                                              180                           GACGGGTACG                                                                             ATATCTCCGA                                                                             CTACTACCAG                                                                             ATCCTCCCCG                                                                             TCCACGGGAC                                                                             CCTGGAGGAC                                                                              240                           TTCACCGTGG                                                                             ACGAGGCCCA                                                                             CGGCCGGGGG                                                                             ATGAAGGTGA                                                                             TCATTGAGCT                                                                             CGTCCTGAAC                                                                              300                           CACACCTCCA                                                                             TTGACCACCC                                                                             TTGGTTCCAG                                                                             GAGGCGAGGA                                                                             AGCCGAATAG                                                                             CCCCATGCGG                                                                              360                           GACTGGTACG                                                                             TGTGGAGCGA                                                                             CACCCCGGAG                                                                             AAGTACAAGG                                                                             GGGTCCGGGT                                                                             CATCTTCAAG                                                                              420                           GACTTTGAAA                                                                             CCTCCAACTG                                                                             GACCTTTGAC                                                                             CCCGTGGCCA                                                                             AGGCCTACTA                                                                             CTGGCACCGC                                                                              480                           TTCTACTGGC                                                                             ACCAGCCCGA                                                                             CCTCAACTGG                                                                             GACAGCCCCG                                                                             AGGTGGAGAA                                                                             GGCCATCCAC                                                                              540                           CAGGTCATGT                                                                             TCTTCTGGGC                                                                             CGACCTGGGG                                                                             GTGGACGGCT                                                                             TCCGCCTGGA                                                                             CGCCATCCCC                                                                              600                           TACCTCTACG                                                                             AGCGGGAGGG                                                                             GACCTCCTGC                                                                             GAGAACCTCC                                                                             CCGAGACCAT                                                                             TGAGGCGGTG                                                                              660                           AAGCGCCTGA                                                                             GGAAGGCCCT                                                                             GGAGGAGCGC                                                                             TACGGCCCCG                                                                             GGAAGATCCT                                                                             CCTCGCCGAG                                                                              720                           GCCAACATGT                                                                             GGCCGGAGGA                                                                             GACCCTCCCC                                                                             TACTTCGGGG                                                                             ACGGGGACGG                                                                             GGTCCACATG                                                                              780                           GCCTACAACT                                                                             TCCCCCTGAT                                                                             GCCCCGGATC                                                                             TTCATGGCCC                                                                             TAAGGCGGGA                                                                             GGACCGGGGT                                                                              840                           CCCATTGAAA                                                                             CCATGCTCAA                                                                             GGAGGCGGAG                                                                             GGGATCCCCG                                                                             AAACCGCCCA                                                                             GTGGGCCCTC                                                                              900                           TTCCTCCGCA                                                                             ACCACGACGA                                                                             GCTCACCCTG                                                                             GAGAAGGTCA                                                                             CGGAGGAGGA                                                                             GCGGGAGTTC                                                                              960                           ATGTACGAGG                                                                             CCTACGCCCC                                                                             CGACCCCAAG                                                                             TTCCGCATCA                                                                             ACCTGGGGAT                                                                             CCGCCGCCGC                                                                             1020                           CTCATGCCCC                                                                             TCCTCGGGGG                                                                             CGACCGCAGG                                                                             CGGTACGAGC                                                                             TCCTCACCGC                                                                             CCTCCTCCTC                                                                             1080                           ACCCTAAAGG                                                                             GCACGCCCAT                                                                             CGTCTACTAC                                                                             GGGGACGAGA                                                                             TCGGCATGGG                                                                             GGACAACCCC                                                                             1140                           TTCCTCGGGG                                                                             ACCGGAACGG                                                                             TGTCAGGACC                                                                             CCCATGCAGT                                                                             GGTCCCAAGA                                                                             CCGCATCGTC                                                                             1200                           GCCTTCTCCC                                                                             GCGCCCCCTA                                                                             CCACGCCCTC                                                                             TTCCTTCCCC                                                                             CCGTGAGCGA                                                                             GGGGCCCTAC                                                                             1260                           AGCTACCACT                                                                             TCGTCAACGT                                                                             GGAGGCCCAG                                                                             CGGGAAAACC                                                                             CCCACTCCCT                                                                             CCTGAGCTTC                                                                             1320                           AACCGCCGCT                                                                             TCCTCGCCCT                                                                             GAGGAACCAG                                                                             CACGCCAAGA                                                                             TCTTCGGCCG                                                                             GGGGAGCCTC                                                                             1380                           ACCCTTCTCC                                                                             CCGTGGAGAA                                                                             CCGGCGCGTC                                                                             CTCGCCTACC                                                                             TGAGGGAGCA                                                                             CGAGGGGGAG                                                                             1440                           CGGGTCCTGG                                                                             TGGTGGCCAA                                                                             CCTCTCCCGC                                                                             TACACCCAGG                                                                             CCTTTGACCT                                                                             CCCCTTGGAG                                                                             1500                           GCCTACCAAG                                                                             GCCTCGTCCC                                                                             CGTGGAGCTC                                                                             TTCTCGCAGC                                                                             AACCCTTCCC                                                                             CCCGGTGGAG                                                                             1560                           GGGCGCTACC                                                                             GCTTGACCCT                                                                             GGGCCCCCAC                                                                             GGCTTCGCCC                                                                             TCTTCGCCCT                                                                             GAAGCCCGTG                                                                             1620                           GAGGCGGTGC                                                                             TCCACCTCCC                                                                             CTCCCCCGAC                                                                             TGGGCCGAGG                                                                             AGCCCGCCCC                                                                             CGAGGAGGCC                                                                             1680                           GACCTGCCCC                                                                             GGGTCCACAT                                                                             GCCCGGGGGG                                                                             CCGGAGGTCC                                                                             TCCTGGTGGA                                                                             CACCCTGGTC                                                                             1740                           CACGAAAGGG                                                                             GGCGGGAGGA                                                                             GCTCCTAAAC                                                                             GCCCTCGCCC                                                                             AGACCCTGAA                                                                             GGAGAAGAGC                                                                             1800                           TGGCTCGCCC                                                                             TCAAGCCGCA                                                                             GAAGGTGGCC                                                                             CTCCTGGACG                                                                             CCCTCCGCTT                                                                             CCAGAAGGAC                                                                             1860                           CCGCCCCTTT                                                                             ACCTCACCCT                                                                             GCTCCAGCTG                                                                             GAGAACCACA                                                                             GGACCCTCCA                                                                             GGTCTCCCTC                                                                             1920                           CCCCTCCTCT                                                                             GGTCCCCCCA                                                                             GAGGCGGGAA                                                                             GGCCCCGGCC                                                                             TCTTCGCCCG                                                                             CACCCACGGC                                                                             1980                           CAGCCCGGCT                                                                             ACTTCTACGA                                                                             GCTCTCCTTG                                                                             GACCCAGGCT                                                                             TCTACCGCCT                                                                             CCTCCTCGCC                                                                             2040                           CGCCTTAAGG                                                                             AGGGGTTTGA                                                                             GGGGCGGAGC                                                                             CTCCGGGCCT                                                                             ACTACCGCGG                                                                             CCGCCACCCG                                                                             2100                           GGTCCCGTGC                                                                             CCGAGGCCGT                                                                             GGACCTCCTC                                                                             CGGCCGGGAC                                                                             TCGCGGCGGG                                                                             GGAGGGGGTC                                                                             2160                           TGGGTCCAGC                                                                             TCGGCCTCGT                                                                             CCAAGACGGG                                                                             GGCCTGGACC                                                                             GCACGGAGCG                                                                             GGTCCTCCCC                                                                             2220                           CGCCTGGACC                                                                             TCCCCTGGGT                                                                             TCTCCGGCCC                                                                             GAAGGGGGCC                                                                             TCTTCTGGGA                                                                             GCGGGGCGCC                                                                             2280                           TCCAGAAGGG                                                                             TCCTCGCCCT                                                                             CACGGGAAGC                                                                             CTCCCCCCGG                                                                             GCCGCCCCCA                                                                             GGACCTCTTC                                                                             2340                           GCCGCCCTGG                                                                             AGGTCCGGCT                                                                             CCTGGAAAGC                                                                             CTTCCCCGCC                                                                             TCCGGGGGCA                                                                             CGCCCCCGGG                                                                             2400                           ACCCCAGGCC                                                                             TCCTTCCCGG                                                                             GGCCCTGCAC                                                                             GAGACCGAAG                                                                             CCCTGGTCCG                                                                             CCTCCTCGGG                                                                             2460                           GTGCGCCTCG                                                                             CCCTCCTCCA                                                                             CCGGGCCCTT                                                                             GGGGAGGTGG                                                                             AGGGGGTGGT                                                                             GGGGGGCCAC                                                                             2520                           CCCCTCCTAG                                                                             GCCGCGGCCT                                                                             CGGGGCCTTC                                                                             CTGGAGCTGG                                                                             AGGGGGAGGT                                                                             GTACCTCGTG                                                                             2580                           GCCCTGGGCG                                                                             CGGAAAAGCG                                                                             GGGCACGGTG                                                                             GAGGAGGACC                                                                             TGGCCCGCCT                                                                             GGCCTACGAC                                                                             2640                           GTGGAGCGGG                                                                             CCGTGCACCT                                                                             CGCCCTCGAG                                                                             GCCCTGGAGG                                                                             CGGAGCTTTG                                                                             GGCCTTTGCC                                                                             2700                           GAGGAGGTGG                                                                             CCGACCACCT                                                                             CCACGCCGCC                                                                             TTCCTCCAAG                                                                             CCTACCGCTC                                                                             CGCCCTCCCC                                                                             2760                           GAGGAGGCCC                                                                             TGGAGGAGGC                                                                             GGGCTGGACG                                                                             CGGCACATGG                                                                             CCGAGGTGGC                                                                             GGCGGAGCAC                                                                             2820                           CTCCACCGGG                                                                             AGGAAAGGCC                                                                             CGCCCGCAAG                                                                             CGCATCCACG                                                                             AGCGCTGGCA                                                                             GGCCAAGGCC                                                                             2880                           GGAAAAGCC                                       2889                           __________________________________________________________________________

The sequential experimental steps used to reveal the amino acid sequence and the base sequence in SEQ ID NOs:3 and 4 are summarized in the below:

(1) A thermostable enzyme was isolated from a culture of a donor microorganism, highly purified, and determined for its amino acid sequence containing the N-terminus. The purified enzyme was partially hydrolyzed with protease, and from which a peptide fragment was isolated and determined for its amino acid sequence;

(2) Separately, a chromosomal DNA was isolated from a donor microorganism's cell, purified and partially digested with a restriction enzyme to obtain a DNA fragment consisting of about 4,000-8,000 base pairs. The DNA fragment was ligated with a DNA ligase to a plasmid vector, which had been previously cut with a restriction enzyme, to obtain a recombinant DNA;

(3) The recombinant DNA was introduced into a microorganism of the species Escherichia coli to obtain transformants, and from which an objective transformant containing a DNA encoding the thermostable enzyme was selected by the colony hybridization method using an oligonucleotide, as a probe, which had been chemically synthesized based on the aforesaid partial amino acid sequence; and

(4) The recombinant DNA was obtained from the selected transformant and annealed with a primer, followed by allowing a DNA polymerase to act on the resultant to extend the primer, and determining the base sequence of the resultant complementary chain DNA by the dideoxy chain termination method. The comparison of an amino acid sequence, which could be estimated based on the determined base sequence, with the aforesaid amino acid sequence concluded that it encodes the thermostable enzyme.

The following Experiments 3 and 4 concretely illustrate the above items (2) to (4), and the techniques used therein were conventional ones commonly used in this field, for example, those described by J. Sumbruck et al. in "Molecular Cloning A Laboratory Manual", 2nd edition, published by Cold Spring Harbor Laboratory Press (1989).

EXPERIMENT 3 PREPARATION OF RECOMBINANT DNA CONTAINING DNA ENCODING THERMOSTABLE ENZYME, AND TRANSFORMANT

Experiment 3-1

Preparation of chromosomal DNA

A seed culture of Thermus aquaticus (ATCC 33923) was inoculated into nutrient broth medium (pH 7.0), and cultured at 60° C. for 24 hours with a rotary shaker. The cells were separated from the resultant culture by centrifugation, suspended in TES buffer (pH 8.0), admixed with 0.05 w/v % lysozyme, and incubated at 37° C. for 30 min. The resultant was freezed at -80° C. for one hour, admixed with TSS buffer (pH 9.0), heated to 60° C., and further admixed with a mixture solution of TES buffer and phenol, and the resultant solution was chilled with ice, followed by centrifugation to obtain a supernatant. To the supernatant was added 2-fold volumes of cold ethanol, and the precipitated crude chromosomal DNA was collected, suspended in SSC buffer (pH 7.1), admixed with 7.5 μg ribonuclease and 125 μg protease, and incubated at 37° C. for one hour. Thereafter, a mixture solution of chloroform and isoamyl alcohol was added to the reaction mixture to extract the objective chromosomal DNA, and the extract was admixed with cold ethanol, followed by collecting the formed sediment containing the chromosomal DNA. The resultant purified chromosomal DNA was dissolved in SSC buffer (pH 7.1) to give a concentration of about one mg/ml, and the resultant solution was freezed at -80° C.

Experiment 3-2

Preparation of recombinant DNA pBTM22 and transformant BTM22

About one ml of the purified chromosomal DNA obtained in Example 3-1 was placed in a test tube, admixed with about 10 units of Sau 3AI, a restriction enzyme, and enzymatically reacted at 37° C. for about 20 min to partially cleave the chromosomal DNA, followed by recovering a DNA fragment consisting of about 4,000-8,000 base pairs by means of sucrose density-gradient ultracentrifugation. One μg of Bluescript II SK(+), a plasmid vector commercialized by Stratagene Cloning Systems, California, USA, was placed in a test tube, subjected to the action of Bam HI, a restriction enzyme, to completely digest the plasmid vector, admixed with 10 μg of the DNA fragment and 2 units of T4 DNA ligase, and allowed to stand at 4° C. overnight to ligate the DNA fragment to the plasmid vector fragment. To the resultant recombinant DNA was added 30 μl of "Epicurian Coli® XLI-Blue", a competent cell commercialized by Stratagene Cloning Systems, California, USA, Japan, allowed to stand under ice-chilling conditions for 30 min, heated to 42° C., admixed with SOC broth, and incubated at 37° C. for one hour to introduce the recombinant DNA into Escherichia coli.

The resultant transformant was inoculated into agar plate (pH 7.0) containing 50 μg/ml of 5-bromo-4-chloro-3-indolyl-β-galactoside, and cultured at 37° C. for 18 hours, followed by placing a nylon film on the agar plate to fix thereon about 6,000 colonies formed on the agar plate. Based on the amino acid sequence of Trp-Tyr-Lys-Asp-Ala-Val as shown in SEQ ID NO:1 (amino acid residues 5-10), the base sequence of probe 1 represented by the base sequence of 5'-TGGTAYAARGAYGCNGT-3' (SEQ ID NO:7) was chemically synthesized, labelled with ³² p, and hybridized with the colonies of transformants fixed on the nylon film, followed by selecting 5 transformants which had strongly hybridized with the probe 1.

The objective recombinant DNA was selected in usual manner from the 5 transformants, and, in accordance with the method described by E. M. Southern in Journal of Molecular Biology, Vol.98, pp.503-517 (1975), the recombinant DNA was hybridized with probe 2 represented by the base sequence of 5'-AAYATGTGGCCNGARGA-3' (SEQ ID NO:8), which had been chemically synthesized based on the amino acid sequence in SEQ ID NO:2, i.e. Asn-Met-Trp-Pro-Glu-Glu (amino acid residues 7-12), and labelled with ³² p, followed by selecting a recombinant DNA which had strongly hybridized with the probe 2. The recombinant DNA and the transformant thus selected were respectively named "pBTM22" and "BTM22".

The transformant BTM22 was inoculated into L-broth (pH 7.0) containing 100 μg/ml ampicillin, and cultured at 37° C. for 24 hours by a rotary shaker. After completion of the culture, the resultant cells were centrifugally collected from the culture, and treated with conventional alkaline method to ex tract a recombinant DNA from the cells. The extract was in usual ma nner purified and analyzed and revealing that the recombinant DNA pBTM22 consists of about 10,300 base pairs. As is shown in FIG. 5, a fragment containing a DNA, which consists of about 2,900 base pairs and encodes the thermostable enzyme, is located in the downstream near the digested site of Hind III, a restriction enzyme.

Experiment 3-3

Production of recombinant enzyme by transformant BTM22

In 500-ml flasks were placed 100 ml aliquots of a liquid nutrient culture medium (pH 7.0) consisting of 2.0 w/v glucose, 0.5 w/v % peptone, 0.1 w/v % yeast extract, 0.1 w/v % dipotassium hydrogen phosphate, 0.06 w/v % sodium dihydrogen phosphate, 0.05 w/v % magnesium sulfate heptahydrate, 0.5 w/v % calcium carbonate and water, and each flasks was sterilized by heating at 115° C. for 30 min, cooled, admixed with 50 μg/ml ampicillin, and inoculated with the transformant BTM22 obtained in Experiment 3-2, followed by culturing the transformant at 37° C. for 24 hours by a rotary shaker. The resultant culture was treated with an ultrasonic disintegrator to disrupt cells, and the resultant suspension was centrifuged to remove insoluble substances. The supernatant thus obtained was assayed for the enzyme activity and revealing that one L of the culture contained about 800 units of a recombinant enzyme.

As a control, a seed culture of Escherichia coli XLI-Blue or Thermus aquaticus (ATCC 33923) was inoculated in a fresh preparation of the same liquid nutrient culture medium but free of ampicillin, and, in the case of culturing Thermus aquaticus (ATCC 33923), it was cultured and treated similarly as above except that the cultivation temperature was set to 65° C. Assaying the activity of the resultant, one L culture of Thermus aquaticus contained about 350 units of the enzyme, and the yield was significantly lower than that of transformant BTM22. Escherichia coli XLI-Blue used as a host did not form the thermostable enzyme.

Thereafter, the enzyme produced by the transformant BTM22 was purified similarly as in Experiments 1 and 2, and examined for its physicochemical properties and features. As a result, it was revealed that it has substantially the same physicochemical properties as the thermostable enzyme of Thermus aquaticus (ATCC 33923), i.e. it has a molecular weight of about 100,000-110,000 daltons on SDS-PAGE and an isoelectric point of about 3.8-4.8 on isoelectrophoresis, and is not substantially inactivated even when incubated at 80° C. for 60 min in water (pH 7.0). The results indicate that the present thermostable enzyme can be prepared by recombinant DNA technology, and the yield can be significantly increased thereby.

EXPERIMENT 4 PREPARATION OF COMPLEMENTARY CHAIN DNA AND DETERMINATION FOR ITS BASE SEQUENCE AND AMINO ACID SEQUENCE

Two μg of the recombinant DNA pBTM22 in Experiment 3-2 was placed in a test tube, admixed with 2M aqueous sodium hydroxide solution to effect degeneration, and admixed with an adequate amount of cold ethanol, followed by collecting the formed sediment containing a template DNA and drying the sediment in vacua. To the template DNA were added 50 pmole/ml of a chemically synthesized primer represented by the base sequence of 5'-GTAAAACGACGGCCAGT-3' (SEQ ID NO:9), 10 μl of 40 mM Tris-HCl buffer (pH 7.5) containing 20 mM magnesium chloride and 20 mM sodium chloride, and the mixture was incubated at 65° C. for 2 min to effect annealing and admixed with 2 μl of an aqueous solution containing dATP, dGTP and dTTP in respective amounts of 7.5 μM, 0.5 μl of α-³² P!dCTP (2 mCi/ml), one μl of 0.1M dithiothreitol, and 2 μl of 1.5 units/ml T7 DNA polymerase, followed by incubating the resultant mixture at 25° C. for 5 min to extend the primer from the 5'-terminus to the 3'-terminus. Thus, a complementary chain DNA was formed.

The reaction product containing the complementary chain DNA was divided into four equal parts, to each of which 2.5 μl of 50 mM aqueous sodium chloride solution containing 80 μM dNTP and 8 μM ddATP, ddCTP, ddGTP or ddTTP was added, and the resultant mixture was incubated at 37° C. for 5 min, followed by suspending the reaction by the addition of 4 μl of 98 v/v % aqueous formamide solution containing 20 mM EDTA, 0.05 w/v % bromophenol blue, and 0.05 w/v % xylene cyanol. The reaction mixture was heated with a boiling-water bath for 3 min, and a small portion of which was placed on a 6 w/v % polyacrylamide gel, and electrophoresed by energizing it with a constant voltage of about 2,000 volts to separate DNA fragments, followed by fixing the gel in usual manner, drying it and subjecting the resultant to autoradiography.

Analyses of the DNA fragments separated on the radiogram revealed that the complementary chain DNA contains the base sequence consisting of about 3,600 base pairs in SEQ ID NO:5. An amino acid sequence estimable from the base sequence was as shown in parallel in SEQ ID NO:5, and it was compared with the amino acid sequence containing the N-terminus or the partial amino acid sequences in SEQ ID NOs:1 and 2 and revealing that the amino acid sequence in SEQ ID NO:1 corresponded to that positioning from 1 to 20 in SEQ ID NO:5, and the amino acid sequence in SEQ ID NO:2 corresponded to that positioning from 236 to 250 in SEQ ID NO:5. These results indicate that the present recombinant enzyme has the amino acid sequence in SEQ ID NO:3, and the amino acid sequence (SEQ ID NO:3) of the DNA derived from Thermus aquaticus (ATCC 33923) is encoded by the base sequence in SEQ ID NO:4.

    __________________________________________________________________________     SEQ ID NO:5:                                                                   __________________________________________________________________________     GCCCCTCCCT                                                                             CCCCCAACCG                                                                             GGCCTTCCCG                                                                             TGGGGGGGGG                                                                             GCACAGCCTG                                                                             GAGGAAGGGG                                                                              60                            TGCTCGACGG                                                                             GGAGGTGCGG                                                                             CCCCTCTTGC                                                                             GCCGTGGGCC                                                                             GTGACCCCTT                                                                             GCGGGCCAGG                                                                              120                           CTTCCCTCCT                                                                             ACCCCGGGGT                                                                             GCGGGTGGAG                                                                             GACAAGGGCT                                                                             TCGCCCTGGC                                                                             CCTGCACTAC                                                                              180                           CGGGGGGCGG                                                                             AGGGCGAGGA                                                                             GAAGGCCCGG                                                                             GCCTGCCTCG                                                                             AGGCCTGGCT                                                                             TAAGGCGGTG                                                                              240                           GAGGGGCTCC                                                                             TGGGGGCCTT                                                                             GGGCCTCGAG                                                                             GCCCTCCCCG                                                                             GCAAGAGGGT                                                                             CCTGGAGCTC                                                                              300                           AAGCCCAAGG                                                                             GGGTGGACAA                                                                             GGGCCAAGCG                                                                             GTCCTCAGGC                                                                             TCCTCGGACG                                                                             CCACCCGGAC                                                                              360                           CACACCCCCG                                                                             TTTACATCGG                                                                             GGACGACACC                                                                             ACCGACGAGG                                                                             CCGCCTTCCT                                                                             CGCCTTAAGG                                                                              420                           GGCCGGGGCC                                                                             TCACCTTCAA                                                                             GGTGGGGGAA                                                                             GGCCCCACGG                                                                             CGGCCCAAGG                                                                             CCGGCTCAAG                                                                              480                           GACGTGGAGG                                                                             AGGTCCTGGC                                                                             CTACTTGCAA                                                                             ACCTACCTCC                                                                             GACCCACTAG                                                                             CCTTTAGGCC                                                                              540                           GTG                                                                               GAC                                                                               CCC                                                                               CTC                                                                               TGG                                                                               TAC                                                                               AAG                                                                               GAC                                                                               GCG                                                                               GTG                                                                               ATC                                                                               TAC                                                                               CAG                                                                               CTC                                                                               CAC                                                                               GTC                                                                                588                           Met                                                                               Asp                                                                               Pro                                                                               Leu                                                                               Trp                                                                               Tyr                                                                               Lys                                                                               Asp                                                                               Ala                                                                               Val                                                                               Ile                                                                               Tyr                                                                               Gln                                                                               Leu                                                                               His                                                                               Val                               1           5              10             15                                   CGC                                                                               TCC                                                                               TTC                                                                               TTT                                                                               GAC                                                                               GCC                                                                               AAC                                                                               AAC                                                                               GAC                                                                               GGC                                                                               TAC                                                                               GGG                                                                               GAC                                                                               TTT                                                                               GAG                                                                               GGC                                                                                636                           Arg                                                                               Ser                                                                               Phe                                                                               Phe                                                                               Asp                                                                               Ala                                                                               Asn                                                                               Asn                                                                               Asp                                                                               Gly                                                                               Tyr                                                                               Gly                                                                               Asp                                                                               Phe                                                                               Glu                                                                               Gly                                        20             25             30                                      CTG                                                                               AGG                                                                               CGG                                                                               AAG                                                                               CTT                                                                               CCC                                                                               TAC                                                                               CTG                                                                               GAG                                                                               GAG                                                                               CTC                                                                               GGG                                                                               GTC                                                                               AAC                                                                               ACC                                                                               CTC                                                                                684                           Leu                                                                               Arg                                                                               Arg                                                                               Lys                                                                               Leu                                                                               Pro                                                                               Tyr                                                                               Leu                                                                               Glu                                                                               Glu                                                                               Leu                                                                               Gly                                                                               Val                                                                               Asn                                                                               Thr                                                                               Leu                                     35             40             45                                         TGG                                                                               CTC                                                                               ATG                                                                               CCC                                                                               TTC                                                                               TTC                                                                               CAG                                                                               TCC                                                                               CCC                                                                               TTG                                                                               AGG                                                                               GAC                                                                               GAC                                                                               GGG                                                                               TAC                                                                               GAT                                                                                732                           Trp                                                                               Leu                                                                               Met                                                                               Pro                                                                               Phe                                                                               Phe                                                                               Gln                                                                               Ser                                                                               Pro                                                                               Leu                                                                               Arg                                                                               Asp                                                                               Asp                                                                               Gly                                                                               Tyr                                                                               Asp                                  50             55             60                                            ATC                                                                               TCC                                                                               GAC                                                                               TAC                                                                               TAC                                                                               CAG                                                                               ATC                                                                               CTC                                                                               CCC                                                                               GTC                                                                               CAC                                                                               GGG                                                                               ACC                                                                               CTG                                                                               GAG                                                                               GAC                                                                                780                           Ile                                                                               Ser                                                                               Asp                                                                               Tyr                                                                               Tyr                                                                               Gln                                                                               Ile                                                                               Leu                                                                               Pro                                                                               Val                                                                               His                                                                               Gly                                                                               Thr                                                                               Leu                                                                               Glu                                                                               Asp                               65             70             75             80                                TTC                                                                               ACC                                                                               GTG                                                                               GAC                                                                               GAG                                                                               GCC                                                                               CAC                                                                               GGC                                                                               CGG                                                                               GGG                                                                               ATG                                                                               AAG                                                                               GTG                                                                               ATC                                                                               ATT                                                                               GAG                                                                                828                           Phe                                                                               Thr                                                                               Val                                                                               Asp                                                                               Glu                                                                               Ala                                                                               His                                                                               Gly                                                                               Arg                                                                               Gly                                                                               Met                                                                               Lys                                                                               Val                                                                               Ile                                                                               Ile                                                                               Glu                                           85             90             95                                   CTC                                                                               GTC                                                                               CTG                                                                               AAC                                                                               CAC                                                                               ACC                                                                               TCC                                                                               ATT                                                                               GAC                                                                               CAC                                                                               CCT                                                                               TGG                                                                               TTC                                                                               CAG                                                                               GAG                                                                               GCG                                                                                876                           Leu                                                                               Val                                                                               Leu                                                                               Asn                                                                               His                                                                               Thr                                                                               Ser                                                                               Ile                                                                               Asp                                                                               His                                                                               Pro                                                                               Trp                                                                               Phe                                                                               Gln                                                                               Glu                                                                               Ala                                        100            105            110                                     AGG                                                                               AAG                                                                               CCG                                                                               AAT                                                                               AGC                                                                               CCC                                                                               ATG                                                                               CGG                                                                               GAC                                                                               TGG                                                                               TAC                                                                               GTG                                                                               TGG                                                                               AGC                                                                               GAC                                                                               ACC                                                                                924                           Arg                                                                               Lys                                                                               Pro                                                                               Asn                                                                               Ser                                                                               Pro                                                                               Met                                                                               Arg                                                                               Asp                                                                               Trp                                                                               Tyr                                                                               Val                                                                               Trp                                                                               Ser                                                                               Asp                                                                               Thr                                     115            120            125                                        CCG                                                                               GAG                                                                               AAG                                                                               TAC                                                                               AAG                                                                               GGG                                                                               GTC                                                                               CGG                                                                               GTC                                                                               ATC                                                                               TTC                                                                               AAG                                                                               GAC                                                                               TTT                                                                               GAA                                                                               ACC                                                                                972                           Pro                                                                               Glu                                                                               Lys                                                                               Tyr                                                                               Lys                                                                               Gly                                                                               Val                                                                               Arg                                                                               Val                                                                               Ile                                                                               Phe                                                                               Lys                                                                               Asp                                                                               Phe                                                                               Glu                                                                               Thr                                  130            135            140                                           TCC                                                                               AAC                                                                               TGG                                                                               ACC                                                                               TTT                                                                               GAC                                                                               CCC                                                                               GTG                                                                               GCC                                                                               AAG                                                                               GCC                                                                               TAC                                                                               TAC                                                                               TGG                                                                               CAC                                                                               CGC                                                                               1020                           Ser                                                                               Asn                                                                               Trp                                                                               Thr                                                                               Phe                                                                               Asp                                                                               Pro                                                                               Val                                                                               Ala                                                                               Lys                                                                               Ala                                                                               Tyr                                                                               Tyr                                                                               Trp                                                                               His                                                                               Arg                               145            150            155            160                               TTC                                                                               TAC                                                                               TGG                                                                               CAC                                                                               CAG                                                                               CCC                                                                               GAC                                                                               CTC                                                                               AAC                                                                               TGG                                                                               GAC                                                                               AGC                                                                               CCC                                                                               GAG                                                                               GTG                                                                               GAG                                                                               1068                           Phe                                                                               Tyr                                                                               Trp                                                                               His                                                                               Gln                                                                               Pro                                                                               Asp                                                                               Leu                                                                               Asn                                                                               Trp                                                                               Asp                                                                               Ser                                                                               Pro                                                                               Glu                                                                               Val                                                                               Glu                                           165            170            175                                  AAG                                                                               GCC                                                                               ATC                                                                               CAC                                                                               CAG                                                                               GTC                                                                               ATG                                                                               TTC                                                                               TTC                                                                               TGG                                                                               GCC                                                                               GAC                                                                               CTG                                                                               GGG                                                                               GTG                                                                               GAC                                                                               1116                           Lys                                                                               Ala                                                                               Ile                                                                               His                                                                               Gln                                                                               Val                                                                               Met                                                                               Phe                                                                               Phe                                                                               Trp                                                                               Ala                                                                               Asp                                                                               Leu                                                                               Gly                                                                               Val                                                                               Asp                                        180            185            190                                     GGC                                                                               TTC                                                                               CGC                                                                               CTG                                                                               GAC                                                                               GCC                                                                               ATC                                                                               CCC                                                                               TAC                                                                               CTC                                                                               TAC                                                                               GAG                                                                               CGG                                                                               GAG                                                                               GGG                                                                               ACC                                                                               1164                           Gly                                                                               Phe                                                                               Arg                                                                               Leu                                                                               Asp                                                                               Ala                                                                               Ile                                                                               Pro                                                                               Tyr                                                                               Leu                                                                               Tyr                                                                               Glu                                                                               Arg                                                                               Glu                                                                               Gly                                                                               Thr                                     195            200            205                                        TCC                                                                               TGC                                                                               GAG                                                                               AAC                                                                               CTC                                                                               CCC                                                                               GAG                                                                               ACC                                                                               ATT                                                                               GAG                                                                               GCG                                                                               GTG                                                                               AAG                                                                               CGC                                                                               CTG                                                                               AGG                                                                               1212                           Ser                                                                               Cys                                                                               Glu                                                                               Asn                                                                               Leu                                                                               Pro                                                                               Glu                                                                               Thr                                                                               Ile                                                                               Glu                                                                               Ala                                                                               Val                                                                               Lys                                                                               Arg                                                                               Leu                                                                               Arg                                  210            215            220                                           AAG                                                                               GCC                                                                               CTG                                                                               GAG                                                                               GAG                                                                               CGC                                                                               TAC                                                                               GGC                                                                               CCC                                                                               GGG                                                                               AAG                                                                               ATC                                                                               CTC                                                                               CTC                                                                               GCC                                                                               GAG                                                                               1260                           Lys                                                                               Ala                                                                               Leu                                                                               Glu                                                                               Glu                                                                               Arg                                                                               Tyr                                                                               Gly                                                                               Pro                                                                               Gly                                                                               Lys                                                                               Ile                                                                               Leu                                                                               Leu                                                                               Ala                                                                               Glu                               225            230            235            240                               GCC                                                                               AAC                                                                               ATG                                                                               TGG                                                                               CCG                                                                               GAG                                                                               GAG                                                                               ACC                                                                               CTC                                                                               CCC                                                                               TAC                                                                               TTC                                                                               GGG                                                                               GAC                                                                               GGG                                                                               GAC                                                                               1308                           Ala                                                                               Asn                                                                               Met                                                                               Trp                                                                               Pro                                                                               Glu                                                                               Glu                                                                               Thr                                                                               Leu                                                                               Pro                                                                               Tyr                                                                               Phe                                                                               Gly                                                                               Asp                                                                               Gly                                                                               Asp                                           245            250            255                                  GGG                                                                               GTC                                                                               CAC                                                                               ATG                                                                               GCC                                                                               TAC                                                                               AAC                                                                               TTC                                                                               CCC                                                                               CTG                                                                               ATG                                                                               CCC                                                                               CGG                                                                               ATC                                                                               TTC                                                                               ATG                                                                               1356                           Gly                                                                               Val                                                                               His                                                                               Met                                                                               Ala                                                                               Tyr                                                                               Asn                                                                               Phe                                                                               Pro                                                                               Leu                                                                               Met                                                                               Pro                                                                               Arg                                                                               Ile                                                                               Phe                                                                               Met                                        260            265            270                                     GCC                                                                               CTA                                                                               AGG                                                                               CGG                                                                               GAG                                                                               GAC                                                                               CGG                                                                               GGT                                                                               CCC                                                                               ATT                                                                               GAA                                                                               ACC                                                                               ATG                                                                               CTC                                                                               AAG                                                                               GAG                                                                               1404                           Ala                                                                               Leu                                                                               Arg                                                                               Arg                                                                               Glu                                                                               Asp                                                                               Arg                                                                               Gly                                                                               Pro                                                                               Ile                                                                               Glu                                                                               Thr                                                                               Met                                                                               Leu                                                                               Lys                                                                               Glu                                     275            280            285                                        GCG                                                                               GAG                                                                               GGG                                                                               ATC                                                                               CCC                                                                               GAA                                                                               ACC                                                                               GCC                                                                               CAG                                                                               TGG                                                                               GCC                                                                               CTC                                                                               TTC                                                                               CTC                                                                               CGC                                                                               AAC                                                                               1452                           Ala                                                                               Glu                                                                               Gly                                                                               Ile                                                                               Pro                                                                               Glu                                                                               Thr                                                                               Ala                                                                               Gln                                                                               Trp                                                                               Ala                                                                               Leu                                                                               Phe                                                                               Leu                                                                               Arg                                                                               Asn                                  290            295            300                                           CAC                                                                               GAC                                                                               GAG                                                                               CTC                                                                               ACC                                                                               CTG                                                                               GAG                                                                               AAG                                                                               GTC                                                                               ACG                                                                               GAG                                                                               GAG                                                                               GAG                                                                               CGG                                                                               GAG                                                                               TTC                                                                               1500                           His                                                                               Asp                                                                               Glu                                                                               Leu                                                                               Thr                                                                               Leu                                                                               Glu                                                                               Lys                                                                               Val                                                                               Thr                                                                               Glu                                                                               Glu                                                                               Glu                                                                               Arg                                                                               Glu                                                                               Phe                               305            310            315            320                               ATG                                                                               TAC                                                                               GAG                                                                               GCC                                                                               TAC                                                                               GCC                                                                               CCC                                                                               GAC                                                                               CCC                                                                               AAG                                                                               TTC                                                                               CGC                                                                               ATC                                                                               AAC                                                                               CTG                                                                               GGG                                                                               1548                           Met                                                                               Tyr                                                                               Glu                                                                               Ala                                                                               Tyr                                                                               Ala                                                                               Pro                                                                               Asp                                                                               Pro                                                                               Lys                                                                               Phe                                                                               Arg                                                                               Ile                                                                               Asn                                                                               Leu                                                                               Gly                                           325            330            335                                  ATC                                                                               CGC                                                                               CGC                                                                               CGC                                                                               CTC                                                                               ATG                                                                               CCC                                                                               CTC                                                                               CTC                                                                               GGG                                                                               GGC                                                                               GAC                                                                               CGC                                                                               AGG                                                                               CGG                                                                               TAC                                                                               1596                           Ile                                                                               Arg                                                                               Arg                                                                               Arg                                                                               Leu                                                                               Met                                                                               Pro                                                                               Leu                                                                               Leu                                                                               Gly                                                                               Gly                                                                               Asp                                                                               Arg                                                                               Arg                                                                               Arg                                                                               Tyr                                        340            345            350                                     GAG                                                                               CTC                                                                               CTC                                                                               ACC                                                                               GCC                                                                               CTC                                                                               CTC                                                                               CTC                                                                               ACC                                                                               CTA                                                                               AAG                                                                               GGC                                                                               ACG                                                                               CCC                                                                               ATC                                                                               GTC                                                                               1644                           Glu                                                                               Leu                                                                               Leu                                                                               Thr                                                                               Ala                                                                               Leu                                                                               Leu                                                                               Leu                                                                               Thr                                                                               Leu                                                                               Lys                                                                               Gly                                                                               Thr                                                                               Pro                                                                               Ile                                                                               Val                                     355            360            365                                        TAC                                                                               TAC                                                                               GGG                                                                               GAC                                                                               GAG                                                                               ATC                                                                               GGC                                                                               ATG                                                                               GGG                                                                               GAC                                                                               AAC                                                                               CCC                                                                               TTC                                                                               CTC                                                                               GGG                                                                               GAC                                                                               1692                           Tyr                                                                               Tyr                                                                               Gly                                                                               Asp                                                                               Glu                                                                               Ile                                                                               Gly                                                                               Met                                                                               Gly                                                                               Asp                                                                               Asn                                                                               Pro                                                                               Phe                                                                               Leu                                                                               Gly                                                                               Asp                                  370            375            380                                           CGG                                                                               AAC                                                                               GGT                                                                               GTC                                                                               AGG                                                                               ACC                                                                               CCC                                                                               ATG                                                                               CAG                                                                               TGG                                                                               TCC                                                                               CAA                                                                               GAC                                                                               CGC                                                                               ATC                                                                               GTC                                                                               1740                           Arg                                                                               Asn                                                                               Gly                                                                               Val                                                                               Arg                                                                               Thr                                                                               Pro                                                                               Met                                                                               Gln                                                                               Trp                                                                               Ser                                                                               Gln                                                                               Asp                                                                               Arg                                                                               Ile                                                                               Val                               385            390            395            400                               GCC                                                                               TTC                                                                               TCC                                                                               CGC                                                                               GCC                                                                               CCC                                                                               TAC                                                                               CAC                                                                               GCC                                                                               CTC                                                                               TTC                                                                               CTT                                                                               CCC                                                                               CCC                                                                               GTG                                                                               AGC                                                                               1788                           Ala                                                                               Phe                                                                               Ser                                                                               Arg                                                                               Ala                                                                               Pro                                                                               Tyr                                                                               His                                                                               Ala                                                                               Leu                                                                               Phe                                                                               Leu                                                                               Pro                                                                               Pro                                                                               Val                                                                               Ser                                           405            410            415                                  GAG                                                                               GGG                                                                               CCC                                                                               TAC                                                                               AGC                                                                               TAC                                                                               CAC                                                                               TTC                                                                               GTC                                                                               AAC                                                                               GTG                                                                               GAG                                                                               GCC                                                                               CAG                                                                               CGG                                                                               GAA                                                                               1836                           Glu                                                                               Gly                                                                               Pro                                                                               Tyr                                                                               Ser                                                                               Tyr                                                                               His                                                                               Phe                                                                               Val                                                                               Asn                                                                               Val                                                                               Glu                                                                               Ala                                                                               Gln                                                                               Arg                                                                               Glu                                        420            425            430                                     AAC                                                                               CCC                                                                               CAC                                                                               TCC                                                                               CTC                                                                               CTG                                                                               AGC                                                                               TTC                                                                               AAC                                                                               CGC                                                                               CGC                                                                               TTC                                                                               CTC                                                                               GCC                                                                               CTG                                                                               AGG                                                                               1884                           Asn                                                                               Phe                                                                               His                                                                               Ser                                                                               Leu                                                                               Leu                                                                               Ser                                                                               Phe                                                                               Asn                                                                               Arg                                                                               Arg                                                                               Phe                                                                               Leu                                                                               Ala                                                                               Leu                                                                               Arg                                     435            440            445                                        AAC                                                                               CAG                                                                               CAC                                                                               GCC                                                                               AAG                                                                               ATC                                                                               TTC                                                                               GGC                                                                               CGG                                                                               GGG                                                                               AGC                                                                               CTC                                                                               ACC                                                                               CTT                                                                               CTC                                                                               CCC                                                                               1932                           Asn                                                                               Gln                                                                               His                                                                               Ala                                                                               Lys                                                                               Ile                                                                               Phe                                                                               Gly                                                                               Arg                                                                               Gly                                                                               Ser                                                                               Leu                                                                               Thr                                                                               Leu                                                                               Leu                                                                               Pro                                  450            455            460                                           GTG                                                                               GAG                                                                               AAC                                                                               CGG                                                                               CGC                                                                               GTC                                                                               CTC                                                                               GCC                                                                               TAC                                                                               CTG                                                                               AGG                                                                               GAG                                                                               CAC                                                                               GAG                                                                               GGG                                                                               GAG                                                                               1980                           Val                                                                               Glu                                                                               Asn                                                                               Arg                                                                               Arg                                                                               Val                                                                               Leu                                                                               Ala                                                                               Tyr                                                                               Leu                                                                               Arg                                                                               Glu                                                                               His                                                                               Glu                                                                               Gly                                                                               Glu                               465            470            475            480                               CGG                                                                               GTC                                                                               CTG                                                                               GTG                                                                               GTG                                                                               GCC                                                                               AAC                                                                               CTC                                                                               TCC                                                                               CGC                                                                               TAC                                                                               ACC                                                                               CAG                                                                               GCC                                                                               TTT                                                                               GAC                                                                               2028                           Arg                                                                               Val                                                                               Leu                                                                               Val                                                                               Val                                                                               Ala                                                                               Asn                                                                               Leu                                                                               Ser                                                                               Arg                                                                               Tyr                                                                               Thr                                                                               Gln                                                                               Ala                                                                               Phe                                                                               Asp                                           485            490            495                                  CTC                                                                               CCC                                                                               TTG                                                                               GAG                                                                               GCC                                                                               TAC                                                                               CAA                                                                               GGC                                                                               CTC                                                                               GTC                                                                               CCC                                                                               GTG                                                                               GAG                                                                               CTC                                                                               TTC                                                                               TCG                                                                               2076                           Leu                                                                               Pro                                                                               Leu                                                                               Glu                                                                               Ala                                                                               Tyr                                                                               Gln                                                                               Gly                                                                               Leu                                                                               Val                                                                               Pro                                                                               Val                                                                               Glu                                                                               Leu                                                                               Phe                                                                               Ser                                        500            505            510                                     CAG                                                                               CAA                                                                               CCC                                                                               TTC                                                                               CCC                                                                               CCG                                                                               GTG                                                                               GAG                                                                               GGG                                                                               CGC                                                                               TAC                                                                               CGC                                                                               TTG                                                                               ACC                                                                               CTG                                                                               GGC                                                                               2124                           Gln                                                                               Gln                                                                               Pro                                                                               Phe                                                                               Pro                                                                               Pro                                                                               Val                                                                               Glu                                                                               Gly                                                                               Arg                                                                               Tyr                                                                               Arg                                                                               Leu                                                                               Thr                                                                               Leu                                                                               Gly                                     515            520            525                                        CCC                                                                               CAC                                                                               GGC                                                                               TTC                                                                               GCC                                                                               CTC                                                                               TTC                                                                               GCC                                                                               CTG                                                                               AAG                                                                               CCC                                                                               GTG                                                                               GAG                                                                               GCG                                                                               GTG                                                                               CTC                                                                               2172                           Pro                                                                               His                                                                               Gly                                                                               Phe                                                                               Ala                                                                               Leu                                                                               Phe                                                                               Ala                                                                               Leu                                                                               Lys                                                                               Pro                                                                               Val                                                                               Glu                                                                               Ala                                                                               Val                                                                               Leu                                  530            535            540                                           CAC                                                                               CTC                                                                               CCC                                                                               TCC                                                                               CCC                                                                               GAC                                                                               TGG                                                                               GCC                                                                               GAG                                                                               GAG                                                                               CCC                                                                               GCC                                                                               CCC                                                                               GAG                                                                               GAG                                                                               GCC                                                                               2220                           His                                                                               Leu                                                                               Pro                                                                               Ser                                                                               Pro                                                                               Asp                                                                               Trp                                                                               Ala                                                                               Glu                                                                               Glu                                                                               Pro                                                                               Ala                                                                               Pro                                                                               Glu                                                                               Glu                                                                               Ala                               545            550            555            560                               GAC                                                                               CTG                                                                               CCC                                                                               CGG                                                                               GTC                                                                               CAC                                                                               ATG                                                                               CCC                                                                               GGG                                                                               GGG                                                                               CCG                                                                               GAG                                                                               GTC                                                                               CTC                                                                               CTG                                                                               GTG                                                                               2268                           Asp                                                                               Leu                                                                               Pro                                                                               Arg                                                                               Val                                                                               His                                                                               Met                                                                               Pro                                                                               Gly                                                                               Gly                                                                               Pro                                                                               Glu                                                                               Val                                                                               Leu                                                                               Leu                                                                               Val                                           565            570            575                                  GAC                                                                               ACC                                                                               CTG                                                                               GTC                                                                               CAC                                                                               GAA                                                                               AGG                                                                               GGG                                                                               CGG                                                                               GAG                                                                               GAG                                                                               CTC                                                                               CTA                                                                               AAC                                                                               GCC                                                                               CTC                                                                               2316                           Asp                                                                               Thr                                                                               Leu                                                                               Vla                                                                               His                                                                               Glu                                                                               Arg                                                                               Gly                                                                               Arg                                                                               Glu                                                                               Glu                                                                               Leu                                                                               Leu                                                                               Asn                                                                               Ala                                                                               Leu                                        580            585            590                                     GCC                                                                               CAG                                                                               ACC                                                                               CTG                                                                               AAG                                                                               GAG                                                                               AAG                                                                               AGC                                                                               TGG                                                                               CTC                                                                               GCC                                                                               CTC                                                                               AAG                                                                               CCG                                                                               CAG                                                                               AAG                                                                               2364                           Ala                                                                               Gln                                                                               Thr                                                                               Leu                                                                               Lys                                                                               Glu                                                                               Lys                                                                               Ser                                                                               Trp                                                                               Leu                                                                               Ala                                                                               Leu                                                                               Lys                                                                               Pro                                                                               Gln                                                                               Lys                                     595            600            605                                        GTG                                                                               GCC                                                                               CTC                                                                               CTG                                                                               GAC                                                                               GCC                                                                               CTC                                                                               CGC                                                                               TTC                                                                               CAG                                                                               AAG                                                                               GAC                                                                               CCG                                                                               CCC                                                                               CTT                                                                               TAC                                                                               2412                           Val                                                                               Ala                                                                               Leu                                                                               Leu                                                                               Asp                                                                               Ala                                                                               Leu                                                                               Arg                                                                               Phe                                                                               Gln                                                                               Lys                                                                               Asp                                                                               Pro                                                                               Pro                                                                               Lys                                                                               Tyr                                  610            615            620                                           CTC                                                                               ACC                                                                               CTG                                                                               CTC                                                                               CAG                                                                               CTG                                                                               GAG                                                                               AAC                                                                               CAC                                                                               AGG                                                                               ACC                                                                               CTC                                                                               CAG                                                                               GTC                                                                               TCC                                                                               CTC                                                                               2460                           Leu                                                                               Thr                                                                               Leu                                                                               Leu                                                                               Gln                                                                               Leu                                                                               Glu                                                                               Asn                                                                               His                                                                               Arg                                                                               Thr                                                                               Leu                                                                               Gln                                                                               Val                                                                               Ser                                                                               Leu                               625            630            635            640                               CCC                                                                               CTC                                                                               CTC                                                                               TGG                                                                               TCC                                                                               CCC                                                                               CAG                                                                               AGG                                                                               CGG                                                                               GAA                                                                               GGC                                                                               CCC                                                                               GGC                                                                               CTC                                                                               TTC                                                                               GCC                                                                               2508                           Pro                                                                               Leu                                                                               Leu                                                                               Trp                                                                               Ser                                                                               Pro                                                                               Gln                                                                               Arg                                                                               Arg                                                                               Glu                                                                               Gly                                                                               Pro                                                                               Gly                                                                               Leu                                                                               Phe                                                                               Ala                                           645            650            655                                  CGC                                                                               ACC                                                                               CAC                                                                               GGC                                                                               CAG                                                                               CCC                                                                               GGC                                                                               TAC                                                                               TTC                                                                               TAC                                                                               GAG                                                                               CTC                                                                               TCC                                                                               TTG                                                                               GAC                                                                               CCA                                                                               2556                           Arg                                                                               Thr                                                                               His                                                                               Gly                                                                               Gln                                                                               Pro                                                                               Gly                                                                               Tyr                                                                               Phe                                                                               Tyr                                                                               Glu                                                                               Leu                                                                               Ser                                                                               Leu                                                                               Asp                                                                               Pro                                        660            665            670                                     GGC                                                                               TTC                                                                               TAC                                                                               CGC                                                                               CTC                                                                               CTC                                                                               CTC                                                                               GCC                                                                               CGC                                                                               CTT                                                                               AAG                                                                               GAG                                                                               GGG                                                                               TTT                                                                               GAG                                                                               GGG                                                                               2604                           Gly                                                                               Phe                                                                               Tyr                                                                               Arg                                                                               Leu                                                                               Leu                                                                               Leu                                                                               Ala                                                                               Arg                                                                               Leu                                                                               Lys                                                                               Glu                                                                               Gly                                                                               Phe                                                                               Glu                                                                               Gly                                     675            680            685                                        CGG                                                                               AGC                                                                               CTC                                                                               CGG                                                                               GCC                                                                               TAC                                                                               TAC                                                                               CGC                                                                               GGC                                                                               CGC                                                                               CAC                                                                               CCG                                                                               GGT                                                                               CCC                                                                               GTG                                                                               CCC                                                                               2652                           Arg                                                                               Ser                                                                               Leu                                                                               Arg                                                                               Ala                                                                               Tyr                                                                               Tyr                                                                               Arg                                                                               Gly                                                                               Arg                                                                               His                                                                               Pro                                                                               Gly                                                                               Pro                                                                               Val                                                                               Pro                                  690            695            700                                           GAG                                                                               GCC                                                                               GTG                                                                               GAC                                                                               CTC                                                                               CTC                                                                               CGG                                                                               CCG                                                                               GGA                                                                               CTC                                                                               GCG                                                                               GCG                                                                               GGG                                                                               GAG                                                                               GGG                                                                               GTC                                                                               2700                           Glu                                                                               Ala                                                                               Val                                                                               Asp                                                                               Leu                                                                               Leu                                                                               Arg                                                                               Pro                                                                               Gly                                                                               Leu                                                                               Ala                                                                               Ala                                                                               Gly                                                                               Glu                                                                               Gly                                                                               Val                               705            710            715            720                               TGG                                                                               GTC                                                                               CAG                                                                               CTC                                                                               GGC                                                                               CTC                                                                               GTC                                                                               CAA                                                                               GAC                                                                               GGG                                                                               GGC                                                                               CTG                                                                               GAC                                                                               CGC                                                                               ACG                                                                               GAG                                                                               2748                           Trp                                                                               Val                                                                               Gln                                                                               Leu                                                                               Gly                                                                               Leu                                                                               Val                                                                               Gln                                                                               Asp                                                                               Gly                                                                               Gly                                                                               Leu                                                                               Asp                                                                               Arg                                                                               Thr                                                                               Glu                                           725            730            735                                  CGG                                                                               GTC                                                                               CTC                                                                               CCC                                                                               CGC                                                                               CTG                                                                               GAC                                                                               CTC                                                                               CCC                                                                               TGG                                                                               GTT                                                                               CTC                                                                               CGG                                                                               CCC                                                                               GAA                                                                               GGG                                                                               2796                           Arg                                                                               Val                                                                               Leu                                                                               Pro                                                                               Arg                                                                               Leu                                                                               Asp                                                                               Leu                                                                               Pro                                                                               Trp                                                                               Val                                                                               Leu                                                                               Arg                                                                               Pro                                                                               Glu                                                                               Gly                                        740            745            750                                     GGC                                                                               CTC                                                                               TTC                                                                               TGG                                                                               GAG                                                                               CGG                                                                               GGC                                                                               GCC                                                                               TCC                                                                               AGA                                                                               AGG                                                                               GTC                                                                               CTC                                                                               GCC                                                                               CTC                                                                               ACG                                                                               2844                           Gly                                                                               Leu                                                                               Phe                                                                               Trp                                                                               Glu                                                                               Arg                                                                               Gly                                                                               Ala                                                                               Ser                                                                               Arg                                                                               Arg                                                                               Val                                                                               Leu                                                                               Ala                                                                               Leu                                                                               Thr                                     755            760            765                                        GGA                                                                               AGC                                                                               CTC                                                                               CCC                                                                               CCG                                                                               GGC                                                                               CGC                                                                               CCC                                                                               CAG                                                                               GAC                                                                               CTC                                                                               TTC                                                                               GCC                                                                               GCC                                                                               CTG                                                                               GAG                                                                               2892                           Gly                                                                               Ser                                                                               Leu                                                                               Pro                                                                               Pro                                                                               Gly                                                                               Arg                                                                               Pro                                                                               Gln                                                                               Asp                                                                               Leu                                                                               Phe                                                                               Ala                                                                               Ala                                                                               Leu                                                                               Glu                                  770            775            780                                           GTC                                                                               CGG                                                                               CTC                                                                               CTG                                                                               GAA                                                                               AGC                                                                               CTT                                                                               CCC                                                                               CGC                                                                               CTC                                                                               CGG                                                                               GGG                                                                               CAC                                                                               GCC                                                                               CCC                                                                               GGG                                                                               2940                           Val                                                                               Arg                                                                               Leu                                                                               Leu                                                                               Glu                                                                               Ser                                                                               Leu                                                                               Pro                                                                               Arg                                                                               Leu                                                                               Arg                                                                               Gly                                                                               His                                                                               Ala                                                                               Pro                                                                               Gly                               785            790            795            800                               ACC                                                                               CCA                                                                               GGC                                                                               CTC                                                                               CTT                                                                               CCC                                                                               GGG                                                                               GCC                                                                               CTG                                                                               CAC                                                                               GAG                                                                               ACC                                                                               GAA                                                                               GCC                                                                               CTG                                                                               GTC                                                                               2988                           Thr                                                                               Pro                                                                               Gly                                                                               Leu                                                                               Leu                                                                               Pro                                                                               Gly                                                                               Ala                                                                               Leu                                                                               His                                                                               Glu                                                                               Thr                                                                               Glu                                                                               Ala                                                                               Leu                                                                               Val                                           805            810            815                                  CGC                                                                               CTC                                                                               CTC                                                                               GGG                                                                               GTG                                                                               CGC                                                                               CTC                                                                               GCC                                                                               CTC                                                                               CTC                                                                               CAC                                                                               CGG                                                                               GCC                                                                               CTT                                                                               GGG                                                                               GAG                                                                               3036                           Arg                                                                               Leu                                                                               Leu                                                                               Gly                                                                               Val                                                                               Arg                                                                               Leu                                                                               Ala                                                                               Leu                                                                               Leu                                                                               His                                                                               Arg                                                                               Ala                                                                               Leu                                                                               Gly                                                                               Glu                                        820            825            830                                     GTG                                                                               GAG                                                                               GGG                                                                               GTG                                                                               GTG                                                                               GGG                                                                               GGC                                                                               CAC                                                                               CCC                                                                               CTC                                                                               CTA                                                                               GGC                                                                               CGC                                                                               GGC                                                                               CTC                                                                               GGG                                                                               3084                           Val                                                                               Glu                                                                               Gly                                                                               Val                                                                               Val                                                                               Gly                                                                               Gly                                                                               His                                                                               Pro                                                                               Leu                                                                               Leu                                                                               Gly                                                                               Arg                                                                               Gly                                                                               Leu                                                                               Gly                                     835            840            845                                        GCC                                                                               TTC                                                                               CTG                                                                               GAG                                                                               CTG                                                                               GAG                                                                               GGG                                                                               GAG                                                                               GTG                                                                               TAC                                                                               CTC                                                                               GTG                                                                               GCC                                                                               CTG                                                                               GGC                                                                               GCG                                                                               3132                           Ala                                                                               Phe                                                                               Leu                                                                               Glu                                                                               Leu                                                                               Glu                                                                               Gly                                                                               Glu                                                                               Val                                                                               Tyr                                                                               Leu                                                                               Val                                                                               Ala                                                                               Leu                                                                               Gly                                                                               Ala                                  850            855            860                                           GAA                                                                               AAG                                                                               CGG                                                                               GGC                                                                               ACG                                                                               GTG                                                                               GAG                                                                               GAG                                                                               GAC                                                                               CTG                                                                               GCC                                                                               CGC                                                                               CTG                                                                               GCC                                                                               TAC                                                                               GAC                                                                               3180                           Glu                                                                               Lys                                                                               Arg                                                                               Gly                                                                               Thr                                                                               Val                                                                               Glu                                                                               Glu                                                                               Asp                                                                               Leu                                                                               Ala                                                                               Arg                                                                               Leu                                                                               Ala                                                                               Tyr                                                                               Asp                               865            870            875            880                               GTG                                                                               GAG                                                                               CGG                                                                               GCC                                                                               GTG                                                                               CAC                                                                               CTC                                                                               GCC                                                                               CTC                                                                               GAG                                                                               GCC                                                                               CTG                                                                               GAG                                                                               GCG                                                                               GAG                                                                               CTT                                                                               3228                           Val                                                                               Glu                                                                               Arg                                                                               Ala                                                                               Val                                                                               His                                                                               Leu                                                                               Ala                                                                               Leu                                                                               Glu                                                                               Ala                                                                               Leu                                                                               Glu                                                                               Ala                                                                               Glu                                                                               Leu                                           885            890            895                                  TGG                                                                               GCC                                                                               TTT                                                                               GCC                                                                               GAG                                                                               GAG                                                                               GTG                                                                               GCC                                                                               GAC                                                                               CAC                                                                               CTC                                                                               CAC                                                                               GCC                                                                               GCC                                                                               TTC                                                                               CTC                                                                               3276                           Trp                                                                               Ala                                                                               Phe                                                                               Ala                                                                               Glu                                                                               Glu                                                                               Val                                                                               Ala                                                                               Asp                                                                               His                                                                               Leu                                                                               His                                                                               Ala                                                                               Ala                                                                               Phe                                                                               Leu                                        900            905            910                                     CAA                                                                               GCC                                                                               TAC                                                                               CGC                                                                               TCC                                                                               GCC                                                                               CTC                                                                               CCC                                                                               GAG                                                                               GAG                                                                               GCC                                                                               CTG                                                                               GAG                                                                               GAG                                                                               GCG                                                                               GGC                                                                               3324                           Gln                                                                               Ala                                                                               Tyr                                                                               Arg                                                                               Ser                                                                               Ala                                                                               Leu                                                                               Pro                                                                               Glu                                                                               Glu                                                                               Ala                                                                               Leu                                                                               Glu                                                                               Glu                                                                               Ala                                                                               Gly                                     915            920            925                                        TGG                                                                               ACG                                                                               CGG                                                                               CAC                                                                               ATG                                                                               GCC                                                                               GAG                                                                               GTG                                                                               GCG                                                                               GCG                                                                               GAG                                                                               CAC                                                                               CTC                                                                               CAC                                                                               CGG                                                                               GAG                                                                               3372                           Trp                                                                               Thr                                                                               Arg                                                                               His                                                                               Met                                                                               Ala                                                                               Glu                                                                               Val                                                                               Ala                                                                               Ala                                                                               Glu                                                                               His                                                                               Leu                                                                               His                                                                               Arg                                                                               Glu                                  930            935            940                                           GAA                                                                               AGG                                                                               CCC                                                                               GCC                                                                               CGC                                                                               AAG                                                                               CGC                                                                               ATC                                                                               CAC                                                                               GAG                                                                               CGC                                                                               TGG                                                                               CAG                                                                               GCC                                                                               AAG                                                                               GCC                                                                               3420                           Glu                                                                               Arg                                                                               Pro                                                                               Ala                                                                               Arg                                                                               Lys                                                                               Arg                                                                               Ile                                                                               His                                                                               Glu                                                                               Arg                                                                               Trp                                                                               Gln                                                                               Ala                                                                               Lys                                                                               Ala                               945            950            955            960                               GGA                                                                               AAA                                                                               GCC                                       3429                           Gly                                                                               Lys                                                                               Ala                                                                            963                                                                      TAGGCGCCCG                                                                             GTAGCCCTTC                                                                             AGCCCCGGGC                                                                             CACGGGGGCC                                                                             TTGGGGTGGA                                                                             AGACGGCCTC                                                                             3489                           CTCGGGGAGG                                                                             AGGCGGCGCT                                                                             TCTTGGCCCG                                                                             GCGGTAGACG                                                                             GCGTCCCACA                                                                             TGCGGCAGAA                                                                             3549                           GGCGCACACC                                                                             GCCCCCGTGG                                                                             TGGGGTAGCC                                                                             GCACCGCTCG                                                                             CACTCCCTAA                                                                             G       3600                           __________________________________________________________________________

As is described above, the present thermostable enzyme capable of converting maltose into trehalose and vice versa which was found as a result of the present inventors' long-term research, and, unlike conventional enzymes, the enzyme has a specific physicochemical properties. The present invention aims to prepare a recombinant enzyme by means of recombinant DNA technology. Referring the following examples, the process for preparing such a recombinant enzyme, its preparation and uses will be described in detail.

The recombinant enzyme as referred to in the present invention includes those in general which are prepared by recombinant DNA technology and capable of converting maltose into trehalose and vice versa. Usually the present recombinant DNA has a revealed amino acid sequence, e.g. the amino acid sequence in SEQ ID NO:3 or a homologous amino acid to it.

Variants containing amino acid sequences, which are homologous to the amino acid sequence in SEQ ID NO:3, can be prepared by replacing one or more amino acids in SEQ ID NO:3 with other amino acids without alternating the inherent activity of the enzyme. Although even when used the same DNA and it also depends on hosts into which the DNA is introduced, as well as on ingredients and components of nutrient culture media used for culturing transformants, and their cultivation temperature and pH, there may be produced modified enzymes which have the enzymatic activity inherent to the enzyme encoded by the DNA but defect one or more amino acids located in nearness to the N-and/or the C-termini of the amino acid sequence in SEQ ID NO:3, or have one or more amino acids newly added to the N-terminus by the modification of intracellular enzymes of hosts after the DNA expression. Such variants can be included in the present recombinant enzyme as long as they have the desired properties.

The recombinant enzyme according to the present invention can be obtained from cultures of transformants containing the specific DNA. Transformants usable in the present invention can be obtained by introducing into appropriate hosts the base sequence in SEQ ID NO:4, homologous base sequences to it, or complementary base sequences to these base sequences. One or more bases in the above mentioned base sequences may be replaced with other bases by means of the degeneracy of genetic code without alternating the amino acid sequence for which they code. Needless to say, one or more bases in the base sequence, which encodes the enzyme or their variants, can be readily replaced with other bases to allow the DNA to actually express the enzyme production in hosts.

Any DNA derived from natural resources and those artificially synthesized can be used in the present invention as long as they have the aforementioned base sequences. The natural resources of the DNA according to the present invention are, for example, microorganisms of the genus Thermus aquaticus (ATCC 33923) from which a gene, containing the DNA used in the present invention, can be obtained. These microorganisms can be inoculated into nutrient culture media and cultured for about 1-3 days under aerobic conditions, and the resultant cells were collected from cultures and subjected to ultrasonication or treated with a cell-wall lysis enzyme such as lysozyme or β-glucanase to extract genes containing the present DNA. In this case, a proteolytic enzyme such as protease can be used in combination with the cell-wall lysis enzyme, and, in the case of treating the cells with ultrasonication, they may be treated in the presence of a surfactant such as sodium dodecyl sulfate (SDS) or treated with the freezing and thawing method. The objective DNA is obtainable by treating the resultant with phenol extraction, alcohol sedimentation, centrifugation, protease treatment and/or ribonuclease treatment used in general in this field. To artificially synthesize the DNA according to the present invention, it can be chemically synthesized by using the base sequence in SEQ ID NO:3, or can be obtained in plasmid form by inserting a DNA, which encodes the amino acid sequence in SEQ ID NO:4, into an appropriate self-replicable vector to obtain a recombinant DNA, introducing the recombinant DNA into an appropriate host to obtain a transformant, culturing the transformant, separating the proliferated cells from the resultant culture, and collecting plasmids containing the recombinant DNA from the cells.

Such a recombinant DNA, for example, in the form of a recombinant DNA, is usually introduced into hosts. Generally the recombinant DNA contains the aforesaid DNA and a self-replicable vector and can be prepared by conventional method with a relative easiness when the material DNA is in hand. Examples of such a vector are plasmid vectors such as pBR322, pUC18, Bluescript II SK(+), pKK223-3, pUB110, pTZ4, pC194, pHV14, TRp7, TEp7, pBS7, etc.; and phage vectors such as λgt·λC, λgt·λB, ρ11, φ1, φ105, etc. Among these plasmid- and phage-vectors, pBR322, pUC18, Bluescript II SK(+), pKK223-3, λgt·λC and λgt·λB are satisfactorily used in case that the present DNA should be expressed in Escherichia coli, while pUB110, pTZ4, pC194, ρ11, φ1 and φ105 are satisfactorily used to express the DNA in microorganisms of the genus Bacillus. The plasmid vectors pHV14, TRp7, TEp7 and pBS7 are suitably used when the recombinant DNA is allowed to grow in 2 or more types of hosts.

The methods used to insert the present DNA into such vectors in the present invention may be conventional ones generally used in this field. A gene containing the present DNA and a self-replicable vector are first digested by a restriction enzyme and/or ultrasonic disintegrator, then the resultant DNA fragments and vector fragments are ligated. To ligate DNA fragments and vectors, they may be annealed if necessary, then subjected to the action of a DNA ligase in vivo or in vitro. The recombinant DNA thus obtained is replicable without substantial limitation by introducing it into an appropriate host, and culturing the resultant transformant.

The recombinant DNA according to the present invention can be introduced into appropriate host microorganisms including Escherichia Coli and those of the genus Bacillus as well as actinomyces and yeasts. In the case of using Escherichia Coli as a host, it can be cultured in the presence of the recombinant DNA and calcium ion, while in the case of using the microorganisms of the genus Bacillus the competent cell method and the colony hybridization method can be employed. Desired transformants can be cloned by the colony hybridization method or by culturing a variety of transformants in nutrient culture media containing either maltose or trehalose and selecting transformants which form trehalose or maltose.

The transformants thus obtained extracellularly produce the objective enzyme when cultured in nutrient culture media. Generally, liquid media in general supplemented with carbon sources, nitrogen sources and/or minerals, and, if necessary, further supplemented with a small amount of amino acids and/or vitamins can be used as the nutrient culture media. Examples of the carbon sources are saccharides such as starch, starch hydrolysate, glucose, fructose and sucrose. Examples of the nitrogen sources are organic- and inorganic-substances containing nitrogen such as ammonia, ammonium salts, urea, nitrate, peptone, yeast extract, defatted soy been, corn steep liquor and beef extract. Cultures containing the objective enzyme can be obtained by inoculating the transformants into nutrient culture media, and incubating them at a temperature of 20°-50° C. and a pH of 2-9 for about 1-6 days under aerobic conditions by aeration-agitation, etc. Such cultures can be used intact as a crude enzyme preparation, and, usually, cells in the cultures can be disrupted with ultrasonic disintegrator and/or cell-wall lysis enzymes prior to use, followed by separating the enzyme from intact cells and cell debris by filtration and/or centrifugation, and purifying the enzyme. The methods used for purifying the enzyme in the invention include conventional ones in general. From cultures intact cells and cell debris are removed and subjected to one or more methods such as concentration, salting out, dialysis, separately sedimentation, gel filtration chromatography, ion-exchange chromatography, hydrophobic chromatography, affinity chromatography, gel electrophoresis and isoelectrophoresis.

As is described above, the present recombinant thermostable enzyme exerts a distinct activity of forming trehalose or maltose from maltose or trehalose respectively even when allowed to act at a temperature of over 55° C., and such an activity has not been found in conventional enzymes. Trehalose has a mild and high-quality sweetness and it has a great advantage of being capable of sweetening food products without fear of causing unsatisfactorily coloration and deterioration because it has no reducing residue within the molecule. By using these properties of the present recombinant thermostable enzyme, maltose, which could not have been used in some field due to its reducibility, can be converted into useful trehalose with a satisfactory handleability and substantial no reducibility.

Explaining now the present enzymatic conversion method in more detail, the wording "maltose" as referred to in the present invention usually means a saccharide composition containing maltose, and any material or method can be used in the present invention as long as trehalose is formed when the present recombinant thermostable enzyme acts thereon or formed thereby. To effectively produce trehalose in an industrial scale, saccharide compositions with a relatively-high maltose content, i.e., usually, about 70 w/w % or more, preferably, about 80 w/w % or more, can be arbitrarily used. Such saccharide compositions can be prepared by conventional methods generally used in this field, for example, those as disclosed in Japanese Patent Publication Nos.11,437/81 and 17,078/81 wherein β-amylase is allowed to act on gelatinized- or liquefied-starch and separating the formed maltose by separation-sedimentation method or dialysis method, or those as disclosed in Japanese Patent Publication Nos.13,089/72 and 3,938/79 wherein β-amylase is allowed to act on gelatinized- or liquefied-starch together with a starch debranching enzyme such as isoamylase or pullulanase.

In the enzymatic conversion method according to the present invention, an effective amount of the present recombinant thermostable enzyme is allowed to coexist in an aqueous medium containing maltose, followed by keeping the resultant mixture at a prescribed temperature and pH to enzymatically react until the desired amount of trehalose is formed. Although the enzymatic reaction proceeds even at a relatively-low concentration of about 0.1 w/w %, d.s.b., the concentration may be set to about 2 w/w % or more, d.s.b., preferably, about 5-50 w/w %, d.s.b., to proceed the enzymatic conversion method in an industrial scale. The reaction temperature and pH are set within the range which effectively forms maltose without inactivating the recombinant enzyme, i.e. a temperature of over 55° C., preferably, about 56°-63° C., and a pH of about 5-10, preferably, about 6-7. The amount of the recombinant enzyme and the reaction time are appropriately set depending on the conditions of the enzymatic reaction. The present enzymatic conversion method effectively converts maltose into trehalose, and the conversion rate reaches up to about 50% or more in some cases.

The reaction mixtures obtainable by the present enzymatic conversion method can be used intact, and, usually, they may be purified prior to use. For example, the reaction mixtures are filtered and centrifuged to remove insoluble substances, and the resultant solutions are decolored with an activated charcoal, desalted and purified with an ion-exchange resin, and concentrated into syrupy products. Depending on use, the syrupy products can be dried in vacuo and spray-dried into solid products. To obtain products substantially consisting of trehalose, the syrupy products are subjected to one or more methods of chromatographies using ion exchangers, activated charcoals or silica gels, fermentation using yeasts, and removal by decomposing reducing saccharides with alkalis. To treat a relatively-large amount of reaction mixtures, ion-exchange chromatographies such as fixed bed-, moving bed-, and pseudo-moving bed-methods as disclosed in Japanese Patent Laid-Open Nos.23,799/83 and 72,598/83 are arbitrarily used in the invention, and these enable the effective and large production of high-trehalose content products which have been difficult to obtain in large quantities.

The trehalose and saccharide compositions containing trehalose thus obtained can be used in a variety of products which should be avoided from the reducibility of saccharide sweeteners, and therefore, they can be arbitrarily used in food products in general, cosmetics and pharmaceuticals as a sweetener, taste-improving agent, quality-improving agent, stability, filler, adjuvant or excipient.

The following examples explain the preparation of the recombinant thermostable enzyme and the enzymatic conversion method of maltose according to the present invention:

EXAMPLE A-1 PREPARATION OF RECOMBINANT ENZYME

To 500-ml Erlenmeyer flasks were added 100 ml aliquots of a nutrient culture medium consisting of 2.0 w/v % glucose, 0.5 w/v % peptone, 0.1 w/v % yeast extract, 0.1 w/v % dipotassium hydrogen phosphate, 0.06 w/v % sodium dihydrogen phosphate, 0.05 w/v % magnesium sulfate heptahydrate, 0.5 w/v % calcium carbonate and water, and each flask was sterilized by heating at 115° C. for 30 min, cooled, admixed with 50 μg/ml ampicillin, and inoculated with the transformant BTM22 obtained in Experiment 1-2, followed by the incubation at 37° C. for 24 hours under rotatory-shaking conditions to obtain a seed culture. To 30-L jar fermenters were added 18 L aliquots of a fresh preparation of the same nutrient culture medium, sterilized similarly as above, admixed with 50 μg/ml ampicillin, and inoculated with 1 v/v % of the seed culture, followed by the incubation at 37° C. and a pH of 6-8 for 24 hours under aeration-agitation conditions. The resultant cultures were pooled, treated with ultrasonication to disrupt cells, centrifuged to remove insoluble substances, followed by assaying the enzymatic activity of the resultant supernatant. As a result, one L of the culture contained about 800 units of the recombinant enzyme. The assay of the supernatant conducted by the method in Experiment 1-1 revealed that in this culture was obtained an about 5 ml aqueous solution containing about 152 units/ml of a recombinant enzyme with a specific activity of about 135 units/mg protein.

EXAMPLE A-2 PREPARATION OF RECOMBINANT THERMOSTABLE ENZYME Example A-2(a)

Preparation of transformant BTM23

Recombinant DNA pBTM22, obtained by the method in Example 3-2, was cleaved with Hind III, a restriction enzyme, to obtain a DNA fragment consisting of about 8,100 base pairs which contain the base sequence positioning from 107 to 2,889 in SEQ ID NO:4.

Eight oligonucleotides containing base sequences represented by 5'-AGCTTGAATTCTTTTTTAATAAAATCAGGAGGAAAAACCATGGA CC-3' (SEQ ID NO:10), 5'-CCCTCTGGTACAAGGACGCGGTGATCTACCAGCTCCAC-3' (SEQ ID NO:10), 5'-GTCCGCT CCTTCTTTGACGCCAACAACGACGGCTACGG-3' (SEQ ID NO:12), 5'-GGACTTTGAGGGCCTGAGG CGGA-3' (SEQ ID NO:13), 5'-AGCTTCCGCCTCAGGCCCTCAAAGTCCCCGTAGCCGTCGTTGTTG-3' (SEQ ID NO:14), 5'-GCGTCAAAGAAGGAGCGGACGTGGAGCTGGTAGATCACC-3' (SEQ ID NO:15), 5'-GCGTCCTTG TACCAGAGGGGGTCCATGGTTTTTCCTCC-3' (SEQ ID NO:16), and 5'-TGATTTTATTAAAAAAGAA TTCA-3 (SEQ ID NO:17) were mixed in adequate amounts, and the mixture was successively incubated at 100° C., 65° C., 37° C. and 20° C. for 20 min, respectively, to anneal the oligonucleotides. A first recombinant DNA, which contains the base sequence in SEQ ID NO:6 and a base sequence consisting of the bases of positions 1-2,889 in SEQ ID NO:3 wherein the guanines (G) located in the positions 1-963 were replaced with adenines (A), was obtained by adding the above DNA fragment to a double stranded DNA of 141 base pairs having 5' cohesive end of 4 bases at each terminus, which consists of the base sequence in SEQ ID NO:6 and the bases of positions 1-110 in SEQ ID NO:4 wherein the guanine (G) located in the position 1 in SEQ ID NO:4 was replaced with adenine (A) without alternating the amino acid sequence consisting of those of positions 1-36 in SEQ ID NO:3, and allowing the mixture to stand at 4° C. overnight in the presence of T4 DNA ligase to anneal the contents.

SEQ ID NO:6:

    AGCTTGAATT CTTTTTTAAT AAAATCAGGA GGAAAAACC                 39

Recombinant DNA pBTM22 obtained by the method in Experiment 3-2 was cleaved with Bam HI, a restriction enzyme, to obtain a DNA fragment consisting of about 2,400 base pairs which contains the base sequence positioning from 1,008 to 2,889 in SEQ ID NO:4 which was then ligated with "M13tv19 RF DNA", a phage vector commercialized by Takara Shuzo Co., Ltd., Tokyo, Japan, which had been cleaved with Bam HI to obtain a second recombinant DNA.

An oligonucleotide containing a base sequence represented by 5'-CGGTAGCCCTGCAGCCCCGGG-3' corresponding to the base sequence positioning at 3,438 to 3,458 in SEQ ID NO:5, where "thymine (T)", the base positioning at 3,448 in SEQ ID NO:5 was replaced with "guanine (G)", was in usual manner chemically synthesized. By using the synthesized oligonucleotide and "MUTAN-G", a site-specific mutation system commercialized by Takara Shuzo Co., Ltd., Tokyo, Japan, a third recombinant DNA, which contained the base sequence positioning from 1,008 to 2,889 bases in SEQ ID NO:4 where "thymine (T)", i.e. the base positioning at 3,448 in SEQ ID NO:5, was replaced with "guanine (G)" without alternating the amino acid sequence positioning from 337 to 963 bases in SEQ ID NO:5 which was contained in the second recombinant DNA, was obtained. The procedure of site-specific mutation followed the manual affixed to the "MUTAN-G".

A DNA fragment, consisting of about 1,390 base pairs containing the base sequence positioning at 1 to 1,358 bases in SEQ ID NO:4 where "guanine (G)", i.e. the first base in SEQ ID NO:4, was replaced with "adenine (A)", obtained by cleaving with restriction enzymes Eco RI and Bgl II, and a DNA fragment consisting of abut 1,550 base pairs containing the base sequence positioning at 1,359 to 2,889 in SEQ ID NO:4 obtained by cleaving the third recombinant DNA with restriction enzymes Bgl II and Pst I, were ligated to "pKK223-3", a plasmid vector commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden, with T4 DNA ligase to obtain the recombinant DNA pBTM23 containing the base sequence in SEQ ID NO:4.

The recombinant DNA pBTM23 thus obtained was introduced into Escherichia coli LE 392 (ATCC 33572) which had been previously prepared into a competent cell according to the method as described by J. Sambrook in "Molecular Cloning, A Laboratory Manual", 2nd edition, pp.1.74-1.81 (1989), published by Cold Spring Harbor Laboratory Press, New York, USA, to obtain the present transformant BTM23 containing the DNA coding for the present enzyme. The transformant was cultured by the method in Experiment 3-2, and the proliferated cells were collected from the resultant culture, and lysed to extract the recombinant DNA which was then purified and analyzed, revealing that the recombinant DNA pBTM23 in FIG. 6 consisted of about 7,500 base pairs and had a DNA fragment containing 2,889 base pairs which was ligated to the downstream of Nco I, a restriction enzyme.

Example A-2(b)

Preparation of recombinant thermostable enzyme using transformant

The transformant BTM23 was cultured similarly as in Example A-1 except that a liquid culture medium (pH 7.0) consisting of one w/v % maltose, 3 w/v % polypeptone, one w/v % "MEAST P1G", a product of Asahi Breweries, Ltd., Tokyo, Japan, 0.1 w/v % sodium dihydrogen phosphate dihydrate, 200 μg/ml ampicillin sodium and water was used. To the resultant culture were added lysozyme from albumen, commercialized by Seikagaku-Kogyo Co., Ltd., Tokyo, Japan, and "TRITON X-100", a surfactant to give respective concentrations of 0.1 mg/ml; and 1 mg/ml, and the resultant was incubated at 37° C. for 16 hours while stirring to extract a recombinant thermostable enzyme from the cells. The suspension was heated at 60° C. for one hour to inactivate concomitant enzymes from Escherichia coli, followed by centrifuging the mixture to remove impurities, and assaying the enzyme activity in the supernatant, revealing that one L culture contained about 120,000 units of the recombinant thermostable enzyme. The supernatant was purified by the method in Experiment 1 to obtain an about 177 ml aqueous solution containing about 1,400 units/ml of the recombinant thermostable enzyme with a specific activity of about 135 units/mg protein.

The properties and features of the purified enzyme were studied by the method Experiment 2, revealing that it has a molecular weight of 100,000-110,000 daltons on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and an isoelectric point of about 3.8-4.8 on isoelectrophoresis, and it is not inactivated even when incubated at 80° C. for 60 min in an aqueous solution (pH 7.0). These physicochemical properties are substantially the same of those of Thermus aquaticus (ATCC 33923) as a donor microorganism.

EXAMPLE B-1 PREPARATION OF TREHALOSE SYRUP BY RECOMBINANT ENZYME

Potato starch powder was suspended in water to give a concentration of 10 w/w %, and the suspension was adjusted to pH 5.5, admixed with 2 units/g starch of "SPITASE HS", an α-amylase specimen commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, and heated at 95° C. to effect gelatinization and liquefaction. Thereafter, the resultant liquefied solution was autoclaved at 120° C. for 20 min to inactivate the remaining enzyme, promptly cooled to 50° C., adjusted to pH 5.0, admixed with 500 units/g starch of an isoamylase specimen commercialized by Hayashibara Biochemical Laboratories, Inc., Okayama, Japan, and 20 units/g starch of a β-amylase specimen commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, and subjected to an enzymatic reaction at 50° C. for 24 hours to obtain a saccharide solution containing about 92 w/w % maltose, d.s.b. The saccharide solution was heated at 100° C. for 20 min to inactivate the remaining enzyme, cooled to 60° C., adjusted to pH 6.5, admixed with one unit/g starch of the recombinant enzyme obtained in Example A-1, and subjected to an enzymatic reaction for 96 hours. The reaction mixture was heated at 100° C. for 10 min to inactivate the remaining enzyme, cooled, filtered, and, in usual manner, decolored with an activated charcoal, desalted and deionized with an ion-exchange resin, and concentrated to obtain a 70 w/w % syrup in a yield of about 95% to the material starch, d.s.b.

The product contains about 68 w/w % trehalose, d.s.b, and has a relatively-low reducibility because of its DE (dextrose equivalent) 18.4, as well as having a mild sweetness, moderate viscosity and moisture-retaining ability, and these render it arbitrarily useful in a variety of compositions such as food products, cosmetics and pharmaceuticals as a sweetener, taste-improving agent, stabilizer, filler, adjuvant or excipient.

EXAMPLE B-2 PREPARATION OF TREHALOSE POWDER BY RECOMBINANT DNA

The reaction mixture obtained in Example B-1 was adjusted to pH 5.0, admixed with 10 units/g starch of "GLUCOZYME", a glucoamylase specimen commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, and subjected to an enzymatic reaction at 50° C. for 24 hours. The reaction mixture thus obtained was heated to inactivate the remaining enzyme, and, in usual manner, decolored, desalted, purified and subjected to i on- exchange column c h romatography using "XT-1016 (polymerization degree of 4%)", a cation exchange resin in Na⁺ -form commercialized by Tokyo Organic Chemical Industries., Ltd., Tokyo, Japan, to increase the trehalose content. More particularly, the ion-exchange resin, previously suspended in water, was packed in 4 jacketed-stainless steel columns with an inner column diameter of 5.4 cm, and the columns were cascaded in series to give a total column length of 20 m. About 5 v/v of the reaction mixture was fed to the columns while the inner column temperature was keeping at 60° C., and fractionated by feeding to the columns with 60° C. hot water at an SV (space velocity) 0.15, followed by collecting high-trehalose content fra ctions. Th e fractions were pooled, and, in usual manner, concentrated, dried in vacuo, and pulverized to obtain a trehalose powder in a yield of about 50% to the material, d.s.b.

The product, which contains about 97 w/w % trehalose, d.s.b, and has a r elatively-low reducing power and a mild sweetness, can be arbitrarily incorporated into a variety of compositions such as food products, cosmetics and pharmaceuticals as a sweetener, taste-improving agent, stabilizer, filler, adjuvant or excipient.

EXAMPLE B-3 PREPARATION OF CRYSTALLINE TREHALOSE POWDER BY RECOMBINANT ENZYME

A high-trehalose content fraction, obtained by the method in Example B-2, was in usual manner decolored with an activated charcoal, desalted with an ion-exchanger, and concentrated into an about 70 w/w % solution. The concentrate was placed in a crystallizer and gradually cooled while stirring to obtain a massecuite with a crystallization percentage of about 45%. The massecuite was sprayed at a pressure of about 150 kg/cm² from a nozzle equipped at the top of a drying tower while about 85° C. hot air was blowing downward from the top of the drying tower, about 45° C. hot air was blowing through under a wire-netting conveyer, which was equipped in the basement of the drying tower, to a crystalline powder collected on the conveyer, and the powder was gradually conveying out from the drying tower. Thereafter, the crystalline powder was transferred to an aging tower and aged for 10 hours in the stream of hot air to complete the crystallization and drying. Thus, a hydrous crystalline trehalose powder was obtained in a yield of about 90% to the material, d.s.b.

The product is substantially free from hygroscopicity and readily handleable, and it can be arbitrarily used in a variety compositions such as food products, cosmetics and pharmaceuticals as a sweetener, taste-improving agent, quality-improving agent, stability, filler, adjuvant or excipient.

EXAMPLE B-4 PREPARATION OF ANHYDROUS CRYSTALLINE TREHALOSE POWDER BY RECOMBINANT ENZYME

A high-trehalose content fraction, obtained by the method in Example B-2, was purified similarly as in Example B-3, and the resultant solution was transferred to a vessel and boiled under a reduced pressure to obtain a syrup with a moisture content of about 3.0 w/w %. The syrup was placed in a crystallizer, admixed with about 1.0 w/w % anhydrous crystalline trehalose as a seed crystal, crystallized at 120° C. while stirring, and transferred to a plain aluminum vessel, followed by aging the contents at 100° C. for 6 hours to form a block. The block thus obtained was pulverized with a cutter, dried by fluidized bed drying to obtain an anhydrous crystalline trehalose powder with a moisture content of about 0.3 w/w % in a yield of about 85% to the material, d.s.b.

The product with a strong dehydrating activity can be arbitrarily used as a desiccant for food products, cosmetics and pharmaceuticals, as well as their materials and intermediates, and also used as a white powdery sweetener with a mild sweetness in food products, cosmetics and pharmaceuticals.

EXAMPLE B-5 PREPARATION OF TREHALOSE POWDER BY RECOMBINANT ENZYME

"MALTOSE HHH", a high-purity maltose commercialized by Hayashibara Biochemical Laboratories, Inc., Okayama, Japan, was dissolved in water to give a concentration of 40 w/w %, heated to 57° C., adjusted to pH 6.5, mixed with 2 units/g maltose, d.s.b., of a recombinant thermostable enzyme obtained by the method in Example A-2, followed by the enzymatic reaction for 48 hours. The reaction mixture was heated at 100° C. for 10 min to inactivate the remaining enzyme, cooled, filtered, decolored with an activated charcoal in usual manner, desalted and purified with an ion-exchange resin, dried in vacuo, and pulverized to obtain a powdery product containing about 73 w/w % trehalose, d.s.b., in a yield of about 90% to the material maltose, d.s.b.

Although the product has a DE (dextrose equivalent) of 19 which is about 30% of that of maltose, it has the same viscosity as that of maltose, as well as having a mild sweetness and an adequate moisture-retaining ability. Thus, the product can be arbitrarily used as a sweetener, quality-improving agent, stabilizer, filler, adjuvant and excipient in a variety of compositions such as food products, cosmetics and pharmaceuticals.

As is described above, the present invention is based on the finding of a novel thermostable enzyme which forms trehalose or maltose when acts on maltose or trehalose. The present invention aims to explore a way to produce such an enzyme in an industrial scale and in a considerably-high yield by recombinant DNA technology. The enzymatic conversion method using the present recombinant thermostable enzyme converts maltose into a saccharide composition containing trehalose, glucose and/or maltose in a considerably-high yield. Trehalose has a mild and high-quality sweetness, and does not have a reducing residue within the molecule, and because of these it can readily sweeten food products in general without fear of causing unsatisfactory coloration and deterioration. The recombinant enzyme with a revealed amino acid sequence can be used with a greater safety for the preparation of trehalose which is premised to be used in food products.

Therefore, the present invention is a useful invention which exerts the aforesaid significant action and effect as well as giving a great contribution to this field.

While there has been described what is at present considered to be the preferred embodiments of the invention, it will be understood the various modifications may be made therein, and it is intended to cover in the appended claims all such modifications as fall within the true spirit and scope of the invention.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 17                                                  (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:20 amino acids                                                      (B) TYPE:amino acid                                                            (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE:peptide                                                     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:1:                                         MetAspProLeuTrpTyrLysAspAlaValIleTyrGlnLeuHisVal                               151015                                                                         ArgSerPhePhe                                                                   20                                                                             (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:15 amino acids                                                      (B) TYPE:amino acid                                                            (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE:peptide                                                     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:2:                                         IleLeuLeuAlaGluAlaAsnMetTrpProGluGluThrLeuPro                                  151015                                                                         (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:963 amino acids                                                     (B) TYPE:amino acid                                                            (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE:peptide                                                     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:3:                                         MetAspProLeuTrpTyrLysAspAlaValIleTyrGlnLeuHisVal                               151015                                                                         ArgSerPhePheAspAlaAsnAsnAspGlyTyrGlyAspPheGluGly                               202530                                                                         LeuArgArgLysLeuProTyrLeuGluGluLeuGlyValAsnThrLeu                               354045                                                                         TrpLeuMetProPhePheGlnSerProLeuArgAspAspGlyTyrAsp                               505560                                                                         IleSerAspTyrTyrGlnIleLeuProValHisGlyThrLeuGluAsp                               65707580                                                                       PheThrValAspGluAlaHisGlyArgGlyMetLysValIleIleGlu                               859095                                                                         LeuValLeuAsnHisThrSerIleAspHisProTrpPheGlnGluAla                               100105110                                                                      ArgLysProAsnSerProMetArgAspTrpTyrValTrpSerAspThr                               115120125                                                                      ProGluLysTyrLysGlyValArgValIlePheLysAspPheGluThr                               130135140                                                                      SerAsnTrpThrPheAspProValAlaLysAlaTyrTyrTrpHisArg                               145150155160                                                                   PheTyrTrpHisGlnProAspLeuAsnTrpAspSerProGluValGlu                               165170175                                                                      LysAlaIleHisGlnValMetPhePheTrpAlaAspLeuGlyValAsp                               180185190                                                                      GlyPheArgLeuAspAlaIleProTyrLeuTyrGluArgGluGlyThr                               195200205                                                                      SerCysGluAsnLeuProGluThrIleGluAlaValLysArgLeuArg                               210215220                                                                      LysAlaLeuGluGluArgTyrGlyProGlyLysIleLeuLeuAlaGlu                               225230235240                                                                   AlaAsnMetTrpProGluGluThrLeuProTyrPheGlyAspGlyAsp                               245250255                                                                      GlyValHisMetAlaTyrAsnPheProLeuMetProArgIlePheMet                               260265270                                                                      AlaLeuArgArgGluAspArgGlyProIleGluThrMetLeuLysGlu                               275280285                                                                      AlaGluGlyIleProGluThrAlaGlnTrpAlaLeuPheLeuArgAsn                               290295300                                                                      HisAspGluLeuThrLeuGluLysValThrGluGluGluArgGluPhe                               305310315320                                                                   MetTyrGluAlaTyrAlaProAspProLysPheArgIleAsnLeuGly                               325330335                                                                      IleArgArgArgLeuMetProLeuLeuGlyGlyAspArgArgArgTyr                               340345350                                                                      GluLeuLeuThrAlaLeuLeuLeuThrLeuLysGlyThrProIleVal                               355360365                                                                      TyrTyrGlyAspGluIleGlyMetGlyAspAsnProPheLeuGlyAsp                               370375380                                                                      ArgAsnGlyValArgThrProMetGlnTrpSerGlnAspArgIleVal                               385390395400                                                                   AlaPheSerArgAlaProTyrHisAlaLeuPheLeuProProValSer                               405410415                                                                      GluGlyProTyrSerTyrHisPheValAsnValGluAlaGlnArgGlu                               420425430                                                                      AsnProHisSerLeuLeuSerPheAsnArgArgPheLeuAlaLeuArg                               435440445                                                                      AsnGlnHisAlaLysIlePheGlyArgGlySerLeuThrLeuLeuPro                               450455460                                                                      ValGluAsnArgArgValLeuAlaTyrLeuArgGluHisGluGlyGlu                               465470475480                                                                   ArgValLeuValValAlaAsnLeuSerArgTyrThrGlnAlaPheAsp                               485490495                                                                      LeuProLeuGluAlaTyrGlnGlyLeuValProValGluLeuPheSer                               500505510                                                                      GlnGlnProPheProProValGluGlyArgTyrArgLeuThrLeuGly                               515520525                                                                      ProHisGlyPheAlaLeuPheAlaLeuLysProValGluAlaValLeu                               530535540                                                                      HisLeuProSerProAspTrpAlaGluGluProAlaProGluGluAla                               545550555560                                                                   AspLeuProArgValHisMetProGlyGlyProGluValLeuLeuVal                               565570575                                                                      AspThrLeuValHisGluArgGlyArgGluGluLeuLeuAsnAlaLeu                               580585590                                                                      AlaGlnThrLeuLysGluLysSerTrpLeuAlaLeuLysProGlnLys                               595600605                                                                      ValAlaLeuLeuAspAlaLeuArgPheGlnLysAspProProLeuTyr                               610615620                                                                      LeuThrLeuLeuGlnLeuGluAsnHisArgThrLeuGlnValSerLeu                               625630635640                                                                   ProLeuLeuTrpSerProGlnArgArgGluGlyProGlyLeuPheAla                               645650655                                                                      ArgThrHisGlyGlnProGlyTyrPheTyrGluLeuSerLeuAspPro                               660665670                                                                      GlyPheTyrArgLeuLeuLeuAlaArgLeuLysGluGlyPheGluGly                               675680685                                                                      ArgSerLeuArgAlaTyrTyrArgGlyArgHisProGlyProValPro                               690695700                                                                      GluAlaValAspLeuLeuArgProGlyLeuAlaAlaGlyGluGlyVal                               705710715720                                                                   TrpValGlnLeuGlyLeuValGlnAspGlyGlyLeuAspArgThrGlu                               725730735                                                                      ArgValLeuProArgLeuAspLeuProTrpValLeuArgProGluGly                               740745750                                                                      GlyLeuPheTrpGluArgGlyAlaSerArgArgValLeuAlaLeuThr                               755760765                                                                      GlySerLeuProProGlyArgProGlnAspLeuPheAlaAlaLeuGlu                               770775780                                                                      ValArgLeuLeuGluSerLeuProArgLeuArgGlyHisAlaProGly                               785790795800                                                                   ThrProGlyLeuLeuProGlyAlaLeuHisGluThrGluAlaLeuVal                               805810815                                                                      ArgLeuLeuGlyValArgLeuAlaLeuLeuHisArgAlaLeuGlyGlu                               820825830                                                                      ValGluGlyValValGlyGlyHisProLeuLeuGlyArgGlyLeuGly                               835840845                                                                      AlaPheLeuGluLeuGluGlyGluValTyrLeuValAlaLeuGlyAla                               850855860                                                                      GluLysArgGlyThrValGluGluAspLeuAlaArgLeuAlaTyrAsp                               865870875880                                                                   ValGluArgAlaValHisLeuAlaLeuGluAlaLeuGluAlaGluLeu                               885890895                                                                      TrpAlaPheAlaGluGluValAlaAspHisLeuHisAlaAlaPheLeu                               900905910                                                                      GlnAlaTyrArgSerAlaLeuProGluGluAlaLeuGluGluAlaGly                               915920925                                                                      TrpThrArgHisMetAlaGluValAlaAlaGluHisLeuHisArgGlu                               930935940                                                                      GluArgProAlaArgLysArgIleHisGluArgTrpGlnAlaLysAla                               945950955960                                                                   GlyLysAla                                                                      (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:2889 base pairs                                                     (B) TYPE:nucleic acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION:SEQ ID NO:4:                                         GTGGACCCCCTCTGGTACAAGGACGCGGTGATCTACCAGCTCCACGTCCGCTCCTTCTTT60                 GACGCCAACAACGACGGCTACGGGGACTTTGAGGGCCTGAGGCGGAAGCTTCCCTACCTG120                GAGGAGCTCGGGGTCAACACCCTCTGGCTCATGCCCTTCTTCCAGTCCCCCTTGAGGGAC180                GACGGGTACGATATCTCCGACTACTACCAGATCCTCCCCGTCCACGGGACCCTGGAGGAC240                TTCACCGTGGACGAGGCCCACGGCCGGGGGATGAAGGTGATCATTGAGCTCGTCCTGAAC300                CACACCTCCATTGACCACCCTTGGTTCCAGGAGGCGAGGAAGCCGAATAGCCCCATGCGG360                GACTGGTACGTGTGGAGCGACACCCCGGAGAAGTACAAGGGGGTCCGGGTCATCTTCAAG420                GACTTTGAAACCTCCAACTGGACCTTTGACCCCGTGGCCAAGGCCTACTACTGGCACCGC480                TTCTACTGGCACCAGCCCGACCTCAACTGGGACAGCCCCGAGGTGGAGAAGGCCATCCAC540                CAGGTCATGTTCTTCTGGGCCGACCTGGGGGTGGACGGCTTCCGCCTGGACGCCATCCCC600                TACCTCTACGAGCGGGAGGGGACCTCCTGCGAGAACCTCCCCGAGACCATTGAGGCGGTG660                AAGCGCCTGAGGAAGGCCCTGGAGGAGCGCTACGGCCCCGGGAAGATCCTCCTCGCCGAG720                GCCAACATGTGGCCGGAGGAGACCCTCCCCTACTTCGGGGACGGGGACGGGGTCCACATG780                GCCTACAACTTCCCCCTGATGCCCCGGATCTTCATGGCCCTAAGGCGGGAGGACCGGGGT840                CCCATTGAAACCATGCTCAAGGAGGCGGAGGGGATCCCCGAAACCGCCCAGTGGGCCCTC900                TTCCTCCGCAACCACGACGAGCTCACCCTGGAGAAGGTCACGGAGGAGGAGCGGGAGTTC960                ATGTACGAGGCCTACGCCCCCGACCCCAAGTTCCGCATCAACCTGGGGATCCGCCGCCGC1020               CTCATGCCCCTCCTCGGGGGCGACCGCAGGCGGTACGAGCTCCTCACCGCCCTCCTCCTC1080               ACCCTAAAGGGCACGCCCATCGTCTACTACGGGGACGAGATCGGCATGGGGGACAACCCC1140               TTCCTCGGGGACCGGAACGGTGTCAGGACCCCCATGCAGTGGTCCCAAGACCGCATCGTC1200               GCCTTCTCCCGCGCCCCCTACCACGCCCTCTTCCTTCCCCCCGTGAGCGAGGGGCCCTAC1260               AGCTACCACTTCGTCAACGTGGAGGCCCAGCGGGAAAACCCCCACTCCCTCCTGAGCTTC1320               AACCGCCGCTTCCTCGCCCTGAGGAACCAGCACGCCAAGATCTTCGGCCGGGGGAGCCTC1380               ACCCTTCTCCCCGTGGAGAACCGGCGCGTCCTCGCCTACCTGAGGGAGCACGAGGGGGAG1440               CGGGTCCTGGTGGTGGCCAACCTCTCCCGCTACACCCAGGCCTTTGACCTCCCCTTGGAG1500               GCCTACCAAGGCCTCGTCCCCGTGGAGCTCTTCTCGCAGCAACCCTTCCCCCCGGTGGAG1560               GGGCGCTACCGCTTGACCCTGGGCCCCCACGGCTTCGCCCTCTTCGCCCTGAAGCCCGTG1620               GAGGCGGTGCTCCACCTCCCCTCCCCCGACTGGGCCGAGGAGCCCGCCCCCGAGGAGGCC1680               GACCTGCCCCGGGTCCACATGCCCGGGGGGCCGGAGGTCCTCCTGGTGGACACCCTGGTC1740               CACGAAAGGGGGCGGGAGGAGCTCCTAAACGCCCTCGCCCAGACCCTGAAGGAGAAGAGC1800               TGGCTCGCCCTCAAGCCGCAGAAGGTGGCCCTCCTGGACGCCCTCCGCTTCCAGAAGGAC1860               CCGCCCCTTTACCTCACCCTGCTCCAGCTGGAGAACCACAGGACCCTCCAGGTCTCCCTC1920               CCCCTCCTCTGGTCCCCCCAGAGGCGGGAAGGCCCCGGCCTCTTCGCCCGCACCCACGGC1980               CAGCCCGGCTACTTCTACGAGCTCTCCTTGGACCCAGGCTTCTACCGCCTCCTCCTCGCC2040               CGCCTTAAGGAGGGGTTTGAGGGGCGGAGCCTCCGGGCCTACTACCGCGGCCGCCACCCG2100               GGTCCCGTGCCCGAGGCCGTGGACCTCCTCCGGCCGGGACTCGCGGCGGGGGAGGGGGTC2160               TGGGTCCAGCTCGGCCTCGTCCAAGACGGGGGCCTGGACCGCACGGAGCGGGTCCTCCCC2220               CGCCTGGACCTCCCCTGGGTTCTCCGGCCCGAAGGGGGCCTCTTCTGGGAGCGGGGCGCC2280               TCCAGAAGGGTCCTCGCCCTCACGGGAAGCCTCCCCCCGGGCCGCCCCCAGGACCTCTTC2340               GCCGCCCTGGAGGTCCGGCTCCTGGAAAGCCTTCCCCGCCTCCGGGGGCACGCCCCCGGG2400               ACCCCAGGCCTCCTTCCCGGGGCCCTGCACGAGACCGAAGCCCTGGTCCGCCTCCTCGGG2460               GTGCGCCTCGCCCTCCTCCACCGGGCCCTTGGGGAGGTGGAGGGGGTGGTGGGGGGCCAC2520               CCCCTCCTAGGCCGCGGCCTCGGGGCCTTCCTGGAGCTGGAGGGGGAGGTGTACCTCGTG2580               GCCCTGGGCGCGGAAAAGCGGGGCACGGTGGAGGAGGACCTGGCCCGCCTGGCCTACGAC2640               GTGGAGCGGGCCGTGCACCTCGCCCTCGAGGCCCTGGAGGCGGAGCTTTGGGCCTTTGCC2700               GAGGAGGTGGCCGACCACCTCCACGCCGCCTTCCTCCAAGCCTACCGCTCCGCCCTCCCC2760               GAGGAGGCCCTGGAGGAGGCGGGCTGGACGCGGCACATGGCCGAGGTGGCGGCGGAGCAC2820               CTCCACCGGGAGGAAAGGCCCGCCCGCAAGCGCATCCACGAGCGCTGGCAGGCCAAGGCC2880               GGAAAAGCC2889                                                                  (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:3600 base pairs                                                     (B) TYPE:nucleic acid                                                          (C) STRANDEDNESS:DOUBLE                                                        (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE:genomic DNA                                                 (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM:Thermus aquaticus                                                 (B) INDIVIDUAL ISOLATE:ATCC 33923                                              (ix) FEATURE:                                                                  (A) NAME/KEY:5'UTR                                                             (B) LOCATION:1..540                                                            (C) IDENTIFICATION METHOD:E                                                    (A) NAME/KEY:mat peptide                                                       (B) LOCATION:541..3429                                                         (C) IDENTIFICATION METHOD:S                                                    (A) NAME/KEY:3'UTR                                                             (B) LOCATION:3430..3600                                                        (C) IDENTIFICATION METHOD:E                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO:5:                                         GCCCCTCCCTCCCCCAACCGGGCCTTCCCGTGGGGGGGGGGCACAGCCTGGAGGAAGGGG60                 TGCTCGACGGGGAGGTGCGGCCCCTCTTGCGCCGTGGGCCGTGACCCCTTGCGGGCCAGG120                CTTCCCTCCTACCCCGGGGTGCGGGTGGAGGACAAGGGCTTCGCCCTGGCCCTGCACTAC180                CGGGGGGCGGAGGGCGAGGAGAAGGCCCGGGCCTGCCTCGAGGCCTGGCTTAAGGCGGTG240                GAGGGGCTCCTGGGGGCCTTGGGCCTCGAGGCCCTCCCCGGCAAGAGGGTCCTGGAGCTC300                AAGCCCAAGGGGGTGGACAAGGGCCAAGCGGTCCTCAGGCTCCTCGGACGCCACCCGGAC360                CACACCCCCGTTTACATCGGGGACGACACCACCGACGAGGCCGCCTTCCTCGCCTTAAGG420                GGCCGGGGCCTCACCTTCAAGGTGGGGGAAGGCCCCACGGCGGCCCAAGGCCGGCTCAAG480                GACGTGGAGGAGGTCCTGGCCTACTTGCAAACCTACCTCCGACCCACTAGCCTTTAGGCC540                GTGGACCCCCTCTGGTACAAGGACGCGGTGATCTACCAGCTCCACGTC588                            MetAspProLeuTrpTyrLysAspAlaValIleTyrGlnLeuHisVal                               151015                                                                         CGCTCCTTCTTTGACGCCAACAACGACGGCTACGGGGACTTTGAGGGC636                            ArgSerPhePheAspAlaAsnAsnAspGlyTyrGlyAspPheGluGly                               202530                                                                         CTGAGGCGGAAGCTTCCCTACCTGGAGGAGCTCGGGGTCAACACCCTC684                            LeuArgArgLysLeuProTyrLeuGluGluLeuGlyValAsnThrLeu                               354045                                                                         TGGCTCATGCCCTTCTTCCAGTCCCCCTTGAGGGACGACGGGTACGAT732                            TrpLeuMetProPhePheGlnSerProLeuArgAspAspGlyTyrAsp                               505560                                                                         ATCTCCGACTACTACCAGATCCTCCCCGTCCACGGGACCCTGGAGGAC780                            IleSerAspTyrTyrGlnIleLeuProValHisGlyThrLeuGluAsp                               65707580                                                                       TTCACCGTGGACGAGGCCCACGGCCGGGGGATGAAGGTGATCATTGAG828                            PheThrValAspGluAlaHisGlyArgGlyMetLysValIleIleGlu                               859095                                                                         CTCGTCCTGAACCACACCTCCATTGACCACCCTTGGTTCCAGGAGGCG876                            LeuValLeuAsnHisThrSerIleAspHisProTrpPheGlnGluAla                               100105110                                                                      AGGAAGCCGAATAGCCCCATGCGGGACTGGTACGTGTGGAGCGACACC924                            ArgLysProAsnSerProMetArgAspTrpTyrValTrpSerAspThr                               115120125                                                                      CCGGAGAAGTACAAGGGGGTCCGGGTCATCTTCAAGGACTTTGAAACC972                            ProGluLysTyrLysGlyValArgValIlePheLysAspPheGluThr                               130135140                                                                      TCCAACTGGACCTTTGACCCCGTGGCCAAGGCCTACTACTGGCACCGC1020                           SerAsnTrpThrPheAspProValAlaLysAlaTyrTyrTrpHisArg                               145150155160                                                                   TTCTACTGGCACCAGCCCGACCTCAACTGGGACAGCCCCGAGGTGGAG1068                           PheTyrTrpHisGlnProAspLeuAsnTrpAspSerProGluValGlu                               165170175                                                                      AAGGCCATCCACCAGGTCATGTTCTTCTGGGCCGACCTGGGGGTGGAC1116                           LysAlaIleHisGlnValMetPhePheTrpAlaAspLeuGlyValAsp                               180185190                                                                      GGCTTCCGCCTGGACGCCATCCCCTACCTCTACGAGCGGGAGGGGACC1164                           GlyPheArgLeuAspAlaIleProTyrLeuTyrGluArgGluGlyThr                               195200205                                                                      TCCTGCGAGAACCTCCCCGAGACCATTGAGGCGGTGAAGCGCCTGAGG1212                           SerCysGluAsnLeuProGluThrIleGluAlaValLysArgLeuArg                               210215220                                                                      AAGGCCCTGGAGGAGCGCTACGGCCCCGGGAAGATCCTCCTCGCCGAG1260                           LysAlaLeuGluGluArgTyrGlyProGlyLysIleLeuLeuAlaGlu                               225230235240                                                                   GCCAACATGTGGCCGGAGGAGACCCTCCCCTACTTCGGGGACGGGGAC1308                           AlaAsnMetTrpProGluGluThrLeuProTyrPheGlyAspGlyAsp                               245250255                                                                      GGGGTCCACATGGCCTACAACTTCCCCCTGATGCCCCGGATCTTCATG1356                           GlyValHisMetAlaTyrAsnPheProLeuMetProArgIlePheMet                               260265270                                                                      GCCCTAAGGCGGGAGGACCGGGGTCCCATTGAAACCATGCTCAAGGAG1404                           AlaLeuArgArgGluAspArgGlyProIleGluThrMetLeuLysGlu                               275280285                                                                      GCGGAGGGGATCCCCGAAACCGCCCAGTGGGCCCTCTTCCTCCGCAAC1452                           AlaGluGlyIleProGluThrAlaGlnTrpAlaLeuPheLeuArgAsn                               290295300                                                                      CACGACGAGCTCACCCTGGAGAAGGTCACGGAGGAGGAGCGGGAGTTC1500                           HisAspGluLeuThrLeuGluLysValThrGluGluGluArgGluPhe                               305310315320                                                                   ATGTACGAGGCCTACGCCCCCGACCCCAAGTTCCGCATCAACCTGGGG1548                           MetTyrGluAlaTyrAlaProAspProLysPheArgIleAsnLeuGly                               325330335                                                                      ATCCGCCGCCGCCTCATGCCCCTCCTCGGGGGCGACCGCAGGCGGTAC1596                           IleArgArgArgLeuMetProLeuLeuGlyGlyAspArgArgArgTyr                               340345350                                                                      GAGCTCCTCACCGCCCTCCTCCTCACCCTAAAGGGCACGCCCATCGTC1644                           GluLeuLeuThrAlaLeuLeuLeuThrLeuLysGlyThrProIleVal                               355360365                                                                      TACTACGGGGACGAGATCGGCATGGGGGACAACCCCTTCCTCGGGGAC1692                           TyrTyrGlyAspGluIleGlyMetGlyAspAsnProPheLeuGlyAsp                               370375380                                                                      CGGAACGGTGTCAGGACCCCCATGCAGTGGTCCCAAGACCGCATCGTC1740                           ArgAsnGlyValArgThrProMetGlnTrpSerGlnAspArgIleVal                               385390395400                                                                   GCCTTCTCCCGCGCCCCCTACCACGCCCTCTTCCTTCCCCCCGTGAGC1788                           AlaPheSerArgAlaProTyrHisAlaLeuPheLeuProProValSer                               405410415                                                                      GAGGGGCCCTACAGCTACCACTTCGTCAACGTGGAGGCCCAGCGGGAA1836                           GluGlyProTyrSerTyrHisPheValAsnValGluAlaGlnArgGlu                               420425430                                                                      AACCCCCACTCCCTCCTGAGCTTCAACCGCCGCTTCCTCGCCCTGAGG1884                           AsnPheHisSerLeuLeuSerPheAsnArgArgPheLeuAlaLeuArg                               435440445                                                                      AACCAGCACGCCAAGATCTTCGGCCGGGGGAGCCTCACCCTTCTCCCC1932                           AsnGlnHisAlaLysIlePheGlyArgGlySerLeuThrLeuLeuPro                               450455460                                                                      GTGGAGAACCGGCGCGTCCTCGCCTACCTGAGGGAGCACGAGGGGGAG1980                           ValGluAsnArgArgValLeuAlaTyrLeuArgGluHisGluGlyGlu                               465470475480                                                                   CGGGTCCTGGTGGTGGCCAACCTCTCCCGCTACACCCAGGCCTTTGAC2028                           ArgValLeuValValAlaAsnLeuSerArgTyrThrGlnAlaPheAsp                               485490495                                                                      CTCCCCTTGGAGGCCTACCAAGGCCTCGTCCCCGTGGAGCTCTTCTCG2076                           LeuProLeuGluAlaTyrGlnGlyLeuValProValGluLeuPheSer                               500505510                                                                      CAGCAACCCTTCCCCCCGGTGGAGGGGCGCTACCGCTTGACCCTGGGC2124                           GlnGlnProPheProProValGluGlyArgTyrArgLeuThrLeuGly                               515520525                                                                      CCCCACGGCTTCGCCCTCTTCGCCCTGAAGCCCGTGGAGGCGGTGCTC2172                           ProHisGlyPheAlaLeuPheAlaLeuLysProValGluAlaValLeu                               530535540                                                                      CACCTCCCCTCCCCCGACTGGGCCGAGGAGCCCGCCCCCGAGGAGGCC2220                           HisLeuProSerProAspTrpAlaGluGluProAlaProGluGluAla                               545550555560                                                                   GACCTGCCCCGGGTCCACATGCCCGGGGGGCCGGAGGTCCTCCTGGTG2268                           AspLeuProArgValHisMetProGlyGlyProGluValLeuLeuVal                               565570575                                                                      GACACCCTGGTCCACGAAAGGGGGCGGGAGGAGCTCCTAAACGCCCTC2316                           AspThrLeuVlaHisGluArgGlyArgGluGluLeuLeuAsnAlaLeu                               580585590                                                                      GCCCAGACCCTGAAGGAGAAGAGCTGGCTCGCCCTCAAGCCGCAGAAG2364                           AlaGlnThrLeuLysGluLysSerTrpLeuAlaLeuLysProGlnLys                               595600605                                                                      GTGGCCCTCCTGGACGCCCTCCGCTTCCAGAAGGACCCGCCCCTTTAC2412                           ValAlaLeuLeuAspAlaLeuArgPheGlnLysAspProProLysTyr                               610615620                                                                      CTCACCCTGCTCCAGCTGGAGAACCACAGGACCCTCCAGGTCTCCCTC2460                           LeuThrLeuLeuGlnLeuGluAsnHisArgThrLeuGlnValSerLeu                               625630635640                                                                   CCCCTCCTCTGGTCCCCCCAGAGGCGGGAAGGCCCCGGCCTCTTCGCC2508                           ProLeuLeuTrpSerProGlnArgArgGluGlyProGlyLeuPheAla                               645650655                                                                      CGCACCCACGGCCAGCCCGGCTACTTCTACGAGCTCTCCTTGGACCCA2556                           ArgThrHisGlyGlnProGlyTyrPheTyrGluLeuSerLeuAspPro                               660665670                                                                      GGCTTCTACCGCCTCCTCCTCGCCCGCCTTAAGGAGGGGTTTGAGGGG2604                           GlyPheTyrArgLeuLeuLeuAlaArgLeuLysGluGlyPheGluGly                               675680685                                                                      CGGAGCCTCCGGGCCTACTACCGCGGCCGCCACCCGGGTCCCGTGCCC2652                           ArgSerLeuArgAlaTyrTyrArgGlyArgHisProGlyProValPro                               690695700                                                                      GAGGCCGTGGACCTCCTCCGGCCGGGACTCGCGGCGGGGGAGGGGGTC2700                           GluAlaValAspLeuLeuArgProGlyLeuAlaAlaGlyGluGlyVal                               705710715720                                                                   TGGGTCCAGCTCGGCCTCGTCCAAGACGGGGGCCTGGACCGCACGGAG2748                           TrpValGlnLeuGlyLeuValGlnAspGlyGlyLeuAspArgThrGlu                               725730735                                                                      CGGGTCCTCCCCCGCCTGGACCTCCCCTGGGTTCTCCGGCCCGAAGGG2796                           ArgValLeuProArgLeuAspLeuProTrpValLeuArgProGluGly                               740745750                                                                      GGCCTCTTCTGGGAGCGGGGCGCCTCCAGAAGGGTCCTCGCCCTCACG2844                           GlyLeuPheTrpGluArgGlyAlaSerArgArgValLeuAlaLeuThr                               755760765                                                                      GGAAGCCTCCCCCCGGGCCGCCCCCAGGACCTCTTCGCCGCCCTGGAG2892                           GlySerLeuProProGlyArgProGlnAspLeuPheAlaAlaLeuGlu                               770775780                                                                      GTCCGGCTCCTGGAAAGCCTTCCCCGCCTCCGGGGGCACGCCCCCGGG2940                           ValArgLeuLeuGluSerLeuProArgLeuArgGlyHisAlaProGly                               785790795800                                                                   ACCCCAGGCCTCCTTCCCGGGGCCCTGCACGAGACCGAAGCCCTGGTC2988                           ThrProGlyLeuLeuProGlyAlaLeuHisGluThrGluAlaLeuVal                               805810815                                                                      CGCCTCCTCGGGGTGCGCCTCGCCCTCCTCCACCGGGCCCTTGGGGAG3036                           ArgLeuLeuGlyValArgLeuAlaLeuLeuHisArgAlaLeuGlyGlu                               820825830                                                                      GTGGAGGGGGTGGTGGGGGGCCACCCCCTCCTAGGCCGCGGCCTCGGG3084                           ValGluGlyValValGlyGlyHisProLeuLeuGlyArgGlyLeuGly                               835840845                                                                      GCCTTCCTGGAGCTGGAGGGGGAGGTGTACCTCGTGGCCCTGGGCGCG3132                           AlaPheLeuGluLeuGluGlyGluValTyrLeuValAlaLeuGlyAla                               850855860                                                                      GAAAAGCGGGGCACGGTGGAGGAGGACCTGGCCCGCCTGGCCTACGAC3180                           GluLysArgGlyThrValGluGluAspLeuAlaArgLeuAlaTyrAsp                               865870875880                                                                   GTGGAGCGGGCCGTGCACCTCGCCCTCGAGGCCCTGGAGGCGGAGCTT3228                           ValGluArgAlaValHisLeuAlaLeuGluAlaLeuGluAlaGluLeu                               885890895                                                                      TGGGCCTTTGCCGAGGAGGTGGCCGACCACCTCCACGCCGCCTTCCTC3276                           TrpAlaPheAlaGluGluValAlaAspHisLeuHisAlaAlaPheLeu                               900905910                                                                      CAAGCCTACCGCTCCGCCCTCCCCGAGGAGGCCCTGGAGGAGGCGGGC3324                           GlnAlaTyrArgSerAlaLeuProGluGluAlaLeuGluGluAlaGly                               915920925                                                                      TGGACGCGGCACATGGCCGAGGTGGCGGCGGAGCACCTCCACCGGGAG3372                           TrpThrArgHisMetAlaGluValAlaAlaGluHisLeuHisArgGlu                               930935940                                                                      GAAAGGCCCGCCCGCAAGCGCATCCACGAGCGCTGGCAGGCCAAGGCC3420                           GluArgProAlaArgLysArgIleHisGluArgTrpGlnAlaLysAla                               945950955960                                                                   GGAAAAGCC3429                                                                  GlyLysAla                                                                      963                                                                            TAGGCGCCCGGTAGCCCTTCAGCCCCGGGCCACGGGGGCCTTGGGGTGGAAGACGGCCTC3489               CTCGGGGAGGAGGCGGCGCTTCTTGGCCCGGCGGTAGACGGCGTCCCACATGCGGCAGAA3549               GGCGCACACCGCCCCCGTGGTGGGGTAGCCGCACCGCTCGCACTCCCTAAG3600                        (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:39 base pairs                                                       (B) TYPE:nucleic acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION:SEQ ID NO:6:                                         AGCTTGAATTCTTTTTTAATAAAATCAGGAGGAAAAACC39                                      (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        TGGTAYAARGAYGCNGT17                                                            (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        AAYATGTGGCCNGARGA17                                                            (2) INFORMATION FOR SEQ ID NO:9:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                        GTAAAACGACGGCCAGT17                                                            (2) INFORMATION FOR SEQ ID NO:10:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 46 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       AGCTTGAATTCTTTTTTAATAAAATCAGGAGGAAAAACCATGGACC46                               (2) INFORMATION FOR SEQ ID NO:11:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 38 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                       CCCTCTGGTACAAGGACGCGGTGATCTACCAGCTCCAC38                                       (2) INFORMATION FOR SEQ ID NO:12:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 38 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                       GTCCGCTCCTTCTTTGACGCCAACAACGACGGCTACGG38                                       (2) INFORMATION FOR SEQ ID NO:13:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                       GGACTTTGAGGGCCTGAGGCGGA23                                                      (2) INFORMATION FOR SEQ ID NO:14:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 45 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                       AGCTTCCGCCTCAGGCCCTCAAAGTCCCCGTAGCCGTCGTTGTTG45                                (2) INFORMATION FOR SEQ ID NO:15:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 39 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                       GCGTCAAAGAAGGAGCGGACGTGGAGCTGGTAGATCACC39                                      (2) INFORMATION FOR SEQ ID NO:16:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 38 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                       GCGTCCTTGTACCAGAGGGGGTCCATGGTTTTTCCTCC38                                       (2) INFORMATION FOR SEQ ID NO:17:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                       TGATTTTATTAAAAAAGAATTCA23                                                      __________________________________________________________________________ 

We claim:
 1. A recombinant DNA molecule, comprising a nucleotide sequence from nucleotides 541 to 3429 of SEQ ID NO:5 encoding an enzyme derived from microorganism genus Thermus, wherein said enzyme has an activity of converting maltose into trehalose and is stable at a temperature of 55° C.
 2. A replicable recombinant DNA which contains a self-replicable vector and a DNA according to claim
 5. 3. The replicable recombinant DNA of claim 2, wherein said self-replicable vector is plasmid vector Bluescript II SK(+) or pKK223-3.
 4. A transformed host cell which is prepared by introducing into a host cell a replicable recombinant DNA which contains a DNA according to claim
 1. 5. The transformed host cell of claim 4, wherein said self-replicable vector is plasmid vector Bluescript II SK(+) or pKK223-3.
 6. The transformed host cell of claim 4, wherein said host is a microorganism of the species Escherichia coli.
 7. A process for preparing a recombinant enzyme, which comprises:culturing a transformed host cell, prepared by introducing into an appropriate host cell a recombinant DNA containing both a self-replicable vector and a DNA encoding the recombinant enzyme of claim 1, in a nutrient culture medium to form the enzyme; and collecting the formed enzyme from the resultant culture.
 8. The process of claim 7, wherein said self-replicable vector is plasmid vector Bluescript II SK(+) or pKK223-3.
 9. The process of claim 7, wherein said host is a microorganism of the species Escherichia coli.
 10. The process of claim 7, wherein the recombinant enzyme formed in the nutrient culture medium is recovered by a technique selected from the group consisting of one or more of the following: centrifugation, filtration, concentration, sating out, dialysis, separatory sedimentation, ion-exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, gel electrophoresis and isolectrophoresis.
 11. The recombinant DNA molecule according to claim 1, further comprising a self-replicable vector sequence.
 12. A host cell transformed with the recombinant DNA molecule according to claim
 1. 13. The host cell according to claim 12, which is Escherichia coli.
 14. A process for preparing a recombinant enzyme, which is capable of converting maltose into trehalose and is stable at a temperature of 55° C., comprising the steps of:culturing a transformed host cell according to claim 12 in a nutrient culture medium to express and accumulate the recombinant enzyme, and recovering the recombinant enzyme accumulated in the culturing step to prepare a recombinant enzyme.
 15. A method for converting maltose to trehalose, comprising the steps of:introducing a recombinant DNA molecule in accordance with claim 5 into a host microorganism to obtain a recombinant microorganism; culturing said recombinant microorganism to express and accumulate said recombinant enzyme; recovering said expressed and accumulated recombinant enzyme; and subjecting maltose to the action of said recovered recombinant enzyme to convert maltose to trehalose.
 16. The recombinant DNA molecule according to claim 1, which is derived from a thermophilic microorganism.
 17. The recombinant DNA molecule according to claim 1, comprising the nucleotide sequence of SEQ ID NO:5. 